Methods of generating tendon tissue in vitro from connective tissue progenitor cells

ABSTRACT

Methods of generating and expanding proliferative, multipotent connective tissue progenitor cells from adult stem cells are provided. Also provided are methods of generating functional tendon grafts in vitro and bone, cartilage and connective tissues in vivo using the isolated cell preparation of connective tissue progenitor cells.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/087,664 filed on Dec. 2, 2008, which is a National Phase of PCTPatent Application No. PCT/IL2007/000047 having International filingdate of Jan. 11, 2007, which claims the benefit of priority of U.S.Provisional Patent Application Nos. 60/861,081 filed on Nov. 27, 2006,60/861,080 filed on Nov. 27, 2006 and 60/757,864 filed on Jan. 11, 2006.The contents of the above applications are all incorporated herein byreference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to adult tissue-derived connective tissueprogenitor cells (CTPs) which exhibit high proliferation rate andmultipotent differentiation potential which is maintained for at least20 passages, and more particularly, to methods of generating and usingsuch cells for cell based therapy and tissue engineering applications.

Cell-based tissue engineering is an evolving interdisciplinary area thatoffers new opportunities for clinical applications, creating a tool forrepairing and replacing damaged or lost tissues with biologicalsubstitutes. The shortage of organ transplants and the exceeding numberof patients on waiting lists greatly encourage the development of thisfield. The fundamentals of tissue engineering combine cells, bioactivematrices and chemically and biophysically defined in-vitro cultureconditions. For tissue engineering, cells must be easily isolated,sufficient in numbers, with a great proliferation capacity and awell-defined differentiation potential. A number of cell sources havebeen suggested including primary cells and stem cells which are eitherhost- or donor-derived. A wide array of matrices, either biologically orsynthetically designed, are to provide the mechanical cues andthree-dimensional environment, supporting cell attachment, migration,proliferation, differentiation and organization into complex tissues.Controlling stem cell proliferation and differentiation into any desiredcell type requires the identification of chemicals (e.g., hormones andgrowth factors) and/or growth conditions (e.g., static or dynamicculturing conditions), which regulate the differentiation into thedesired cell or tissue.

Connective tissue repair and regeneration are subjected to intensiveresearch within clinical medicine. Damaged or disordered connectivetissues, such as bone, cartilage and tendons need to be reconstructed orreplaced due to traumatic injuries, degenerative diseases, tumorresections and congenital malformations. Current strategies inreconstructive orthopedic surgery include the use of autografts,allografts and artificial substitutes, all subjected to variouslimitations. While the use of cell grafts is limited by availability andmorbidity, synthetic grafts are osteoconductively inferior to theirbiological counterparts, and could fail.

Thus, for cell based therapy and tissue engineering applications,methods of isolating and expanding stem or progenitor cells which cangive rise to an unlimited amount of connective tissue cell lineagescapable of forming connective tissue in vitro are highly desired.

Various studies attempted to identify culturing conditions which can beused to generate connective tissue progenitor cells which exhibitunlimited expansion in culture and differentiation potential to cells ofthe connective tissue lineages.

For example, bone marrow-derived MSCs can be cultured in a culturemedium (e.g., DMEM) supplemented with serum. However, although cellscultured under such conditions and isolated from the first and secondpassage in culture were shown capable of differentiating into theadipocytic, chondrocytic, or osteocytic lineages (see e.g., Pittenger,M. F et al, 1999), the use of such cells in tissue engineeringapplications such as for the in vitro construction of a mature tissuewas never demonstrated.

In another study Sottile, et al., 2002, cultured human trabecularbone-derived cells in a culture medium containing serum and fibroblastgrowth factor (FGF-2). However, the resulting cells reached confluencyonly after 10-20 days, demonstrating their relatively slow proliferationrate. Although cells isolated from the first passage could be induced todifferentiate into osteoblasts, chondrocytes and adipocytes, their lowproliferation rate limits their use in cell based therapy and tissueengineering applications.

In another study Zuk, P. A., et al., 2001, cultured processedlipoaspirate (PLA) cells which were obtained from fat tissues in aculture medium (DMEM) supplemented with serum. However, although thecells could be passaged 13 times, their proliferation rate was extremelyslow as evidenced by the low passaging frequency (i.e., every 12-13days). Thus, although PLA cells from the first passage were capable ofdifferentiating into the adipogenic, chondrogenic, myogenic, andosteogenic lineages, their low proliferation rate limits their use forcell based and tissue engineering applications.

U.S. Pat. Appl. No. 20050260748 discloses a method of isolating adultstem cells from an adipose tissue and culturing them in a mediumcontaining N-acetyl-L-cysteine, an antioxidant (e.g., vitamin C) andnicotinamide. For induction into osteogenic differentiation, the cellswere cultured in a medium containing dexamethasone,L-ascorbate-2-phosphate and beta-glycerphosphate.

In yet another study, Mastrogiacomo, M., et al., 2005, cultured humanskeletal muscle cells in a culture medium containing fibroblast growthfactor (FGF-2) and dexamethason and following two passages in such amedium the cells were further induced to differentiate in vitro into thechondrogenic, osteogenic and adipogenic cell lineages. However, thepotential of using muscle-derived stem cells cultured under suchconditions for generating engineered tissues in vitro was never shown.

Thus, the currently available culturing methods of stem cells do notteach the in vitro construction an engineered tissue (e.g., a maturetissue) in the absence of a scaffold, carrier or a cell support. Forexample, none of the above-described methods enables the formation of amature tendon tissue.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, methods of generating adult stem cells-derivedmultipotent cells which are suitable for tissue engineering devoid ofthe above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of generating connective tissue progenitor cells, comprisingculturing adult stem cells in a culture medium which comprisesdexamethasone and ascorbic acid so as to allow differentiation of theadult stem cells into the connective tissue progenitor cells, whereinthe connective tissue progenitor cells are capable of differentiatinginto at least two cell lineages of a connective tissue; therebygenerating the connective tissue progenitor cells.

According to further features in preferred embodiments of the inventiondescribed below, the method further comprising passaging the connectivetissue progenitor cells in a presence of the culture medium whichcomprises dexamethasone and ascorbic acid, wherein a first passage ofthe passaging is effected no more than 10 days following initialculturing of the adult stem cells in the culture medium.

According to still further features in the described preferredembodiments the at least two cell lineages of the connective tissue areselected from the group consisting of an osteogenic lineage, achondrogenic lineage, an adipocytic lineage and a tendon and ligamentlineage.

According to still further features in the described preferredembodiments the adult stem cells are obtained from a fetal tissue.

According to still further features in the described preferredembodiments the adult stem cells are obtained from a postnatal tissue.

According to still further features in the described preferredembodiments the fetal tissue is obtained from a placenta, amniocytes,amniotic membrane, cord blood and/or chorionic villi sample (CVS).

According to still further features in the described preferredembodiments the postnatal tissue is obtained from umbilical cord, cordblood, foreskin, bone marrow, blood, fat tissue, connective tissueand/or fibroblasts.

According to still further features in the described preferredembodiments the method further comprising expanding the connectivetissue progenitor cells in the culture medium which comprisesdexamethasone and ascorbic acid so as to obtain at least 3.5×10⁹connective tissue progenitor cells from a single cell of the adult stemcells following about 20 passages.

According to still further features in the described preferredembodiments culturing is effected under feeder-free culturingconditions.

According to still further features in the described preferredembodiments the culture medium further comprises inorganic phosphate.

According to still further features in the described preferredembodiments the culture medium further comprises serum or serumreplacement.

According to still further features in the described preferredembodiments the adult tissue stem cells are of a human origin.

According to still further features in the described preferredembodiments passaging is effected every 2-5 days.

According to still further features in the described preferredembodiments passaging is effected for at least 20 times.

According to still further features in the described preferredembodiments culturing is effected under xeno-free conditions.

According to still further features in the described preferredembodiments passaging is effected under xeno-free conditions.

According to still further features in the described preferredembodiments passaging is effected under feeder-free culturingconditions.

According to another aspect of the present invention there is providedan isolated cell preparation of connective tissue progenitor cellsresultant of the method.

According to still further features in the described preferredembodiments the connective tissue progenitor cells are capable of beingmaintained in a proliferative, non-terminally differentiated state forat least 20 passages in culture.

According to still further features in the described preferredembodiments the connective tissue progenitor cells express CD105, CD166,CD44, CD29 and HLA-ABC.

According to still further features in the described preferredembodiments the connective tissue progenitor cells not expressing CD45and HLA-DR.

According to still further features in the described preferredembodiments the isolated cell preparation is devoid of feeder cells.

According to still further features in the described preferredembodiments the isolated cell preparation is xeno-free.

According to still further features in the described preferredembodiments the connective tissue progenitor cells are capable ofdifferentiating into cells of a chondrogenic lineage, an osteogeniclineage, an adipocytic lineage, a tendon and ligament lineage.

According to still further features in the described preferredembodiments cells isolated following the at least 20 passages in cultureare capable of differentiating into cells of a chondrogenic lineage, anosteogenic lineage, an adipocytic lineage, a tendon and ligamentlineage.

According to still further features in the described preferredembodiments the connective tissue progenitor cells are capable offorming mineralized matrix.

According to still further features in the described preferredembodiments the connective tissue progenitor cells are capable offorming a bone tissue.

According to still further features in the described preferredembodiments the connective tissue progenitor cells are capable offorming an extracellular matrix (ECM).

According to still further features in the described preferredembodiments the connective tissue progenitor cells are capable offorming a tendon tissue.

According to still further features in the described preferredembodiments the connective tissue progenitor cells are capable offorming a ligament tissue.

According to still further features in the described preferredembodiments the connective tissue progenitor cells are capable offorming a cartilage tissue.

According to yet another aspect of the present invention there isprovided a method of generating a tendon tissue, the method comprisingculturing the connective tissue progenitor cells of the isolated cellpreparation in a culture medium which comprises ascorbic acid and/ordexamethason under culture conditions devoid of a carrier, therebygenerating the tendon tissue.

According to still another aspect of the present invention there isprovided a method of forming an extracellular matrix (ECM), the methodcomprising culturing the connective tissue progenitor cells of theisolated cell preparation of cells in a culture medium which comprisesascorbic acid, thereby forming the ECM.

According to still further features in the described preferredembodiments the culture medium further comprises dexamethason.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing methods of generating andusing connective tissue progenitor cells from adult stem cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-g are FACS analyses of adult stem cells—derived CTPs(generated from a fetal tissue) showing high level of population puritywith surface markers characteristic of MSCs. Adult stem cells derivedCTPs were cultured in CTP medium for 16 passages and were subjected toFACS analysis using antibodies specific to CD105 (eBioscience, SanDiego, Calif., Cat. No. 12-1057-73) (FIG. 1 a), CD166 (Serotec, Raleigh,N.C., USA, Cat. No. MCA1926F) (FIG. 1 b), CD44 (eBioscience, Cat. No.10-0441-81) (FIG. 1 c), CD29 (Serotec, Cat. No. MCA1926F) (FIG. 1 d),CD45 (Pharmingen, Cat. No. 345809) (FIG. 1 e), HLA-ABC (eBioscience,Cat. No. 12-9983-71) (FIG. 1 f), HLA-DR (eBioscience, Cat. No.12-9956-71) (FIG. 1 g). Note the positive expression of typical MSCsurface markers, including CD105 (FIG. 1 a), CD166 (FIG. 1 b), CD44(FIG. 1 g) and CD29 (FIG. 1 d), and the negative expression (absence) ofCD45 (FIG. 1 e), a hematopoietic marker. Level of cell purity wasconfirmed to be high. Additionally, all cells were positive for HLA-ABC(FIG. 1 f) and negative for HLA-DR (FIG. 1 h, the majorhistocompatibility complex antigens).

FIGS. 2 a-u are RT-PCR analyses depicting the expression of markerscharacteristic of bone, cartilage, tendons and ligaments at passage 1(p1) and passage 10 (p10) of both adult stem cells-derived CTPs(generated from human fetal bone tissue) (lanes 4 and 5, respectively)and hESC-derived CTPs (lanes 2 and 3, respectively), compared toundifferentiated hESCs (u.d., lane 1) and negative (neg., lane 6)control. Adult stem cells derived CTPs were cultured in CTP medium for 1or 10 passages, following which RNA was prepared and RT-PCR reactionswere performed using the PCR primers listed in Table 1 of the Examplessection which follows. The tested markers were: CBFA1 (FIG. 2 a), SOX9(FIG. 2 b), Col-I (Collagen type I, FIG. 2 c), Osteonectin (FIG. 2 d),Osteopontin (FIG. 2 e), ALP (alkaline phosphatase, FIG. 2 f), PTHR1(FIG. 2 g), Col-IX (Collagen type I, FIG. 2 h), Col-X (Collagen type X;FIG. 2 i), COMP (FIG. 2 j), Aggrecan (FIG. 2 k), CS4 (FIG. 2 l),Scleraxis (FIG. 2 m), Col-III (Collagen type III, FIG. 2 n), Col-XII(Collagen type XII, FIG. 2 o), Decorin (FIG. 2 p), Biglycan (FIG. 2 q),Elastin (FIG. 2 r), Fibronectin (FIG. 2 s), Tenascin C (FIG. 2 t) andGAPDH (FIG. 2 u). Note that both human fetal-derived CTPs andhESC-derived CTPs express high levels of core binding factor alpha 1(CBFA1; FIG. 2 a) and SOX9 (FIG. 2 b), both are early transcriptionfactors known to play a major role in osteoblast and chondrocytedifferentiation. Also note that type I collagen (Col-I, FIG. 2 c), themost abundant extracellular protein which is synthesized by osteoblasts,osteonectin (FIG. 2 d) and osteopontin (FIG. 2 e, mainly at passage 1),two major non-collagenous bone matrix proteins, parathyroid hormonereceptor 1 (PTHR1; FIG. 2 g, mainly at passage 1), which regulatesmineral homeostasis and bone formation, and bone-specific alkalinephosphatase (ALP; FIG. 2 f), which binds phosphor to calcium and formsbone hydroxyapetite, are all detected at low and high passages,indicating osteogenic potential. Adult stem cells derived CTPs were alsopositive for cartilage matrix markers: chondroitin sulfate proteoglycan4 (CS4; FIG. 2 l), a cartilage matrix proteoglycan, type X collagen(FIG. 2 i, mainly at passage 1), which is a non-fibril-forming collagenrestricted to the hypertrophic, calcifying zone of growth platecartilage, and cartilage oligomeric matrix protein (COMP; FIG. 2 j,mainly fetal derived CTPs at passage 1), a key non-collagenous cartilagematrix protein. In addition, note the expression of tendon and ligamentspecific markers, such as scleraxis (FIG. 2 m, mainly at passage 10), atranscription factor expressed both in their mature and early progenitorpopulations, and other ECM-related proteins, including type III(Col-III; FIG. 2 n) and type XII (Col-XII; FIG. 2 o) collagens, decorin(FIG. 2 p), biglycan (FIG. 2 q), elastin (FIG. 2 r), fibronectin (FIG. 2s), and tenascin-C (FIG. 2 t), were detected.

FIGS. 3 a-c depict morphology of CTPs derived from connective tissue ofa fetus (FIG. 3 a) and from adult fat tissue (FIGS. 3 b-c); FIG. 3a—CTPs from passage 1; FIG. 3 b—CTPs from passage 1; FIG. 3 c—CTPs frompassage 5;

FIGS. 4 a-c are X-ray images of mice subcutaneously transplanted(ectopic model) with the fat-derived CTPs of the present invention. FIG.4 a—3 weeks post surgery; FIGS. 4 b and c—5 months post surgery; Notethe presence of radio-opaque tissue (arrows) demonstrating the formationof bone tissue in vivo following 3 weeks or 5 months of transplantation.

FIGS. 5 a-f are photomicrographs depicting spontaneous tendon formationby fat tissue-derived CTPs, passage 1. High-density, confluent CTPcultures promote the formation of a few cell wide string-like structure(FIG. 5 a), arising and growing over the surface of the culture plate(FIG. 5 b, focused on the bottom surface of the plate). At laterdevelopmental stages, the structure progressively become wider, longerand well defined, with tendon-like morphology, and elongatedfibroblast-like cells growing in parallel (FIGS. 5 c-f).

FIGS. 5 g-m are photomicrographs depicting spontaneous tendon formationby fetal-derived CTPs, passage 10-14. FIG. 5 g—Immunofluorescenceanalysis of the spontaneously formed tendon tissue using Type-I collagenantibody (Chemicon, Cat. No. MAB3991); FIG. 5 h—A macroscopic view of aspontaneously formed tendon tissue; FIGS. 5 i-j—SEM analyses of thespontaneously formed tendon tissue [magnifications are ×500 (FIG. 5 i)and ×5000 (FIG. 5 j)]; FIG. 5 k, l, m—TEM analysis of the spontaneouslyformed tendon tissue.

FIGS. 6 a-b are photomicrographs depicting differentiation of fattissue-derived CTPs (FIG. 6 a) or fetal-derived CTPs (FIG. 6 b) intoadipocytes. FIG. 6 a—light microscopy of fat-derived CTPs whichdifferentiates into adipocytes. FIG. 6 b—Histological staining (H&Estaining) of fetal derived CTPs which differentiated into adipocytes.

FIGS. 7 a-b are photomicrographs of immunofluorescence analysisdepicting that the adult stem cells derived CTPs of the presentinvention (from fetal tissue) express alkaline phosphatase (ALP; usingthe anti ALP antibody, R&D, Systems Inc, Minneapolis, Minn., USA, Cat.No. MAB1448) (FIG. 7 a) and osteocalcin (using the anti osteocalcinantibody, R&D, Cat. No. MAB1419) (FIG. 7 b). Nuclei were counterstainedwith DAPI (blue). Scale bars, 100 μm.

FIGS. 8 a-d depict the formation of bone matrix from the adult stemcells-derived CTPs. FIG. 8 a—microscopic view of CTP culture (generatedfrom fetal tissue) demonstrating enhanced mineralization after 11passages. FIG. 8 b—Alizarin Red staining of CTP culture (generated fromfetal tissue) confirming the presence of mineralization. FIG. 8c—Immunostaining analysis with type I collagen (Chemicon, Cat. No.MAB3991) demonstrating the presence of self-produced matrix in CTPcultures generated from fetal; FIG. 8 d—SEM analysis of fetal derivedCTPs demonstrating mineralization.

FIGS. 9 a-b depict the in-vitro chondrogenic differentiation of theCTPs. Adult stem cells derived CTPs (taken from any passage of 1-25passages) were cultured in CTP medium for 10-14 days without culturesplitting and were subjected to histological and immunostaininganalyses. FIG. 9 a—Histological examination of intact CTP cultures(generated from a fetal tissue) which were grown in suspension. The H&Estaining showing round chondrocyte-like cells embedded in lacunae, atthe periphery of the sample. Scale bar—100 μm; FIG. 9 b—Picro-sirius redstaining detected collagenous matrix surrounding the cells. Scale bar—20μm.

FIG. 10 is a photomicrograph of a one month old pellet section stainedwith Toluidine blue. Directed differentiation was induced through pelletcultures (passage 9) in the presence of low serum TGF-β3 supplementedmedium. Note the presence of matrix proteoglycans (blue) in the pelletculture. Earlier pellet cultures were not positively stained (data notshown). Scale bar, 100 μm.

FIGS. 11 a-c depict ectopic new bone formation following transplantationof the fat-derived CTPs of the present invention. FIG. 11 a—amacroscopic view of a mouse bearing ectopic subcutaneous transplants offat derived CTPs obtained from any passage of 4-20 passages. Note thevisible ectopic transplants (marked with an arrow, FIG. 11 a) shown 12weeks following transplantation. FIG. 11 b is a macroscopic view of a12-week old ectopic transplant (as described in FIG. 4) followingremoval of skin. Note the round shape (characteristics of anon-cancerous mass), well-vascularized ectopic transplant mass, shownseparated in FIG. 11 c demonstrating that the ectopic transplant of theadult stem cells derived CTPs (generated from fat tissue) isbiocompatible, well integrated within the recipient mouse and notrejected by its immune system.

FIG. 12 a-b are images of bone tissue formed following subcutaneoustransplantation of the fat-derived CTPs (as described in the descriptionof FIGS. 4 and 11). FIG. 12 a—a frozen section of non-demineralizedtissue demonstrating the formation of mineral deposits. Scale bar: 100μm. FIG. 12 b—histological section stained with H&E demonstrating theformation of bone tissue.

FIGS. 13 a-e schematically depict the strategy of repairing criticalAchilles-tendon injury by implanting a tendon graft formed from thehESCs derived CTPs of the present invention. FIG. 13 a—High-densityhESCs derived CTP cultures are grown with no further splitting for 4-5weeks to form sheet-like tissues in culture plates. FIG. 13 b—Oncesheet-like tissues are formed, the tissues are gently removed fromplates using a cell scraper and rolled to form rounded cylinders. FIG.13 c—Non-absorbable sutures are inserted at the ends of a constructthrough all layers. FIG. 13 d—Constructs are immediately used fortransplantation or kept inside custom-made templates made from flexiblesilicon tubes embedded in agar plates. Full thickness, 3-4 mm longsegment of the Achilles tendon in nude mice is cut to form a criticalgap. FIG. 13 e—The tendon graft constructs are sutured to the proximaland distal edges of the injured Achilles tendon.

FIGS. 14 a-b depict the repair of a critical Achilles-tendon injury invivo following transplantation of a tendon which was formed in vitrofrom adult stem cells-derived CTPs (generated from fetal tissue). FIG.14 a—A macroscopic view of a non-transplanted animal which was subjectedto critical Achilles-tendon injury (excision of the Achilles-tendon).Note that the mouse is unable to extent its ankle, resulting in amaximal extension of is less than 90 degrees (yellow lines). FIG. 14 b—amacroscopic view of the in vitro formed tendon graft following itsimplantation into a mouse leg, replacing critically injured Achillestendon.

FIGS. 15 a-f depict macroscopic view (FIG. 15 l) and SEM analyses (FIGS.15 b-f) of freeze dried engineered tissue, derived from fat-CTPs

FIGS. 16 a-f are SEM analyses of cells (293 human embryonic kidneycells) seeded on the freeze dried engineered tissue, derived fromfat-CTPs (FIGS. 16 b, 16 d, 16 f, and the corresponding tissues withoutseeded cells (FIGS. 16 a, 16 c, 16 e), demonstrating thebiocompatibility of the freeze dried engineered tissue.

FIGS. 16 g-h are histological sections of the 293 human embryonic kidneycells seeded on the freeze-dried engineered tissue, derived fromfat-CTPs. FIG. 16 g—low magnification (×100); FIG. 16 h—highmagnification (×400). Note the presence of the 293T epithelial cells andthe CTP-derived ECM (marked by arrows).

FIG. 17 is an image of SEM depicting high magnification of a single 293human embryonic kidney cell seeded on the freeze dried engineered tissueshown in FIGS. 16 a-h, producing nano scale, native ECM.

FIGS. 18 a-f depict acellularized engineered tissue, derived fromfat-CTPs. FIGS. 18 a, b—macroscopic appearance of the acellularizedengineered tissue. FIG. 18 c—H&E staining of acellularized engineeredtissue from fat derived CTPs. Note the absence of nuclei or othercellular components and the presence of fibrous ECM. FIG. 18d—fibronectin immunohistochemical staining [using the anti fibronectinantibody, Diagnostic BioSystems, Inc. (DBS) Pleasanton, Calif., USA,Cat. No. RP013] of the acellularized engineered tissue; FIG. 18 e—Type Icollagen immunofluorescence analysis of the acellularized engineeredtissue; FIG. 18 f—Type II collagen immunofluorescence (using the anticollagen type II antibody, Chemicon, Cat. No. MAB8887) analysis of theacellularized engineered tissue.

FIGS. 18 g-i depict human keratinocyte cells (HACAT cell line) seeded onthe acellularized engineered tissue derived from fat-CTPs one hour(FIGS. 18 g, h) or 5 days (FIG. 18 i) following seeding the cells on theacellularized engineered tissue. FIG. 18 g—SEM analysis of two cellsusing ×3000 magnification (size bar=10 μm); FIG. 18 h—SEM analysis ofmultiple cells using ×1000 magnification (size bar=20 μm). FIG. 18 i—H&Estaining of the seeded cells on the acellularized engineered tissue(magnification ×100). Altogether, these results demonstrate thebiocompatibility of the de-cellularized engineered tissue.

FIGS. 19 a-f depict transplantation of the acellularized tissue(generated from fetal derived CTPs) in vivo. FIGS. 19 a-b theacellularized freeze dried tissue is inserted through an incision in theskin of an ICR mouse. FIGS. 19 c-d—macroscopic appearance of thetransplanted tissue one week following transplantation. FIGS. 19e-f—histological sections of the transplanted tissue one week followingtransplantation stained with H&E.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods of generating connective tissueprogenitor cells (CTPs) from adult stem cells and of using such cellsfor cell based therapy and tissue engineering. Specifically, the presentinvention can be used to repair and regenerate damaged or diseasedtissue by administering the CTPs of the present invention to a subjectin need thereof or by implanting in the subject a tissue graft generatedin vitro by the CTPs of the present invention.

The principles and operation of the method of generating connectivetissue progenitor cells according to the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Cell-based tissue engineering is an evolving interdisciplinary area thatoffers new opportunities for clinical applications, creating a tool forrepairing and replacing damaged or lost tissues with biologicalsubstitutes. The current approaches of repairing damaged or disorderedconnective tissues, such as bone, cartilage and tendons include the useof autografts, allografts and artificial substitutes. However, while theuse of cell grafts is limited by availability and morbidity, syntheticgrafts are osteoconductively inferior to their biological counterparts,and could fail.

Thus, for cell based therapy and tissue engineering applications,methods of isolating and expanding stem or progenitor cells which cangive rise to an unlimited amount of connective tissue cell lineagescapable of forming connective tissue in vitro are highly desired.

Various studies attempted to identify culturing conditions which can beused to generate connective tissue progenitor cells that are capable ofunlimited expansion in culture and which exhibit differentiationpotential to cells of the connective tissue lineages. These include, forexample, culturing of bone marrow-derived MSCs in a culture mediumsupplemented with serum (see e.g., Pittenger, M. F et al, 1999);culturing of bone-derived cells in a culture medium containing serum andfibroblast growth factor (FGF-2) (Sottile, et al., 2002); culturing ofprocessed lipoaspirate (PLA) cells in a culture medium (DMEM)supplemented with serum (Zuk, P. A., et al., 2001); culturing adiposederived stem cells in a medium containing N-acetyl-L-cysteine, anantioxidant (e.g., vitamin C) and nicotinamide (U.S. Pat. Appl. No.20050260748); and culturing of skeletal muscle cells in a culture mediumcontaining fibroblast growth factor (FGF-2) and dexamethason(Mastrogiacomo, M., et al., 2005). However, although cells isolated fromearly passages (e.g., passage 1 or 2) of such culturing conditionsexhibited a differentiation potential into various cell lineages such asthe osteogenic, chondrogenic and adipogenic cell lineages, thedifferentiation potential of such cells from later passages such aspassage 20 was never shown. In addition, some culturing conditions(e.g., those taught by Sottile, et al., 2002 and Zuk, P. A., et al.,2001) resulted in slow proliferation rate of the cells which limitstheir use for therapeutic and pharmaceutical applications. Moreover, theuse of such culturing conditions for the construction of engineeredtissues (e.g., a mature tissue such as a tendon tissue) in the absenceof scaffold or carrier or for cell based therapy was never demonstrated.

While reducing the present invention to practice, the present inventorshave uncovered that culturing of connective tissue progenitor (CTP)cells isolated from adult tissue can be performed in a culture mediumwhich comprises cortisol (e.g., dexamethasone) and ascorbic acid andthat cells cultured in such a culture medium and subject to seriallypassaging can be expanded in vitro for extended periods of time (e.g.,at least 20 passages) and maintain their multipotent differentiationpotential. This is in sharp contrast to other studies which useddexamethason and ascorbic acid in order to induce the terminaldifferentiation of the progenitor cells into the osteogenic lineage (seefor example, Mastrogiacomo, M., et al., 2005; Pittenger, M. F et al,1999; Sottile, et al., 2002; U.S. Pat. Appl. No. 20050260748 and Zuk, P.A., et al., 2001).

As is shown in the Examples section which follows, the CTPs generatedaccording to the method of the present invention are highlyproliferative cells and can be expanded in culture for at least 20passages while maintaining, even following 20 passages, theirdifferentiation potential to any of the connective tissue cell type. Inaddition, as is further shown in the Examples section which follows, theCTPs of the present invention were capable of differentiating into theosteogenic lineage, the chondrogenic lineage, cartilage lineage(cartilage cells), tendon and ligament lineage (tendon cells, ligamentcells) and ECM-forming cells. In addition, CTPs of the present inventionwere capable of forming a functional tendon without using any scaffoldor carrier. Moreover, when transplanted into mice, the CTPs of thepresent invention were capable of forming a loose connective tissue, abone tissue and a cartilage tissue in vivo.

Thus, according to one aspect of the present invention there is provideda method of generating connective tissue progenitor cells. The method iseffected by culturing adult stem cells in a culture medium whichcomprises cortisol and ascorbic acid so as to allow differentiation ofthe adult stem cells into connective tissue progenitor cells, whereinthe connective tissue progenitor cells are capable of differentiatinginto at least two cell lineages of the connective tissue; therebygenerating the connective tissue progenitor cells.

As used herein the phrase “connective tissue progenitor cells (CTPs)”refers to cells which are capable of differentiating to more than onecell lineage and/or cell type of a connective tissue. Examples ofconnective tissues include, but are not limited to dense connectivetissue (e.g., ligament, tendon, periodontal ligament), areolarconnective tissue (e.g., with proteinaceous fibers such as collagen andelastin), reticular connective tissue, adipose tissue, blood, bone,cartilage, skin, intervertebral disc, dental pulp, dentin, gingival,extracellular matrix (ECM)-forming cells, loose connective tissue andsmooth muscle cells.

Preferably, the connective tissue progenitor cells of the presentinvention are capable of differentiating into at least two, morepreferably, at least three, more preferably, at least four cell lineagesor cell types of the osteogenic lineage, a chondrogenic lineage, anadipocytic lineage, tendon cells, ligament cells, areolar connectivetissue and ECM-forming cells.

The phrase “adult stem cells” as used herein refers to cells obtainedfrom a fetal tissue (i.e., taken from a conceptus post implantationuntil birth) or a post natal tissue (i.e., a tissue from a newborn oradult individual) which are capable of differentiation into other celltypes and/or cell lineage. Preferably, the adult stem cells are obtainedfrom a mammal such as primate or human being, preferably, the adult stemcells are derived from a fetal or a post natal human being.

The adult stem cells of the present invention can be isolated fromvarious biological samples which contain adult stem cells of a fetal ora post natal tissue. Non-limiting examples of biological samplescontaining adult stem cells of a fetal tissue which can be used alongwith the method of this aspect of the present invention include aplacental tissue which can be obtained from a placental biopsy of anongoing pregnancy, or following pregnancy termination (using, e.g., asyringe with a needle); chorionic villi sample (CVS) which can beobtained from an ongoing pregnancy (between the 9-14 weeks of gestation)by inserting a catheter through the cervix or a needle into the abdomenand removing a small sample of the placenta (i.e., chorionic villus);amniocytes which can be obtained via amniocentesis of an ongoingpregnancy usually between the 16^(th) to the 20^(th) week of gestation(can be also performed on a later gestational week) by inserting a thinneedle through the abdomen into the uterus; cord blood sample which canbe obtained via cordocentesis of an ongoing pregnancy (at any gestationweek following the 17^(th) week of gestation) by inserting a thin needlethrough the abdomen and uterine walls to the umbilical cord; amnioticmembrane which can be obtained from a terminated pregnancy orimmediately after birth by removal of the amniotic sac.

Non-limiting examples of biological samples containing adult stem cellsof a post-natal tissue which can be used along with the method of thisaspect of the present invention include Wharton's jelly (the matrixpresent within the umbilical cord) and cord blood which can be obtainedfrom the umbilical cord of a newborn individual shortly after birth;foreskin cells (e.g., foreskin fibroblasts) which can be obtained from anewborn or older individual via circumcision; fibroblasts of anyconnective tissue (see for example, Rieske P, Krynska B, Azizi S A.,2005, Differentiation, 73: 474-83); bone marrow which can be obtained atany age using a syringe needle (bone marrow aspirates) from the iliaccrest, femora, tibiae, spine, rib or other medullar spaces; fat tissuewhich can be obtained from subcutaneous, breast or perirenal sites,using liposuction surgery or penniculectomy; connective tissue (e.g.,loose or dense connective tissue, such as bone tissue) which can beobtained using common biopsy obtaining methods.

Prior to culturing, the adult stem cells are preferably isolated fromthe biological sample containing thereof. Methods of isolating adultstem cells are known in the arts and include, for example, thosedisclosed inhttp://www[[.]]dotworthington-biochem[[.]]dotcom/default[[.]]dothtml orprovided along with the Cell Isolation Optimizing System (catalogue no.LK003200; Worthington Biochemical Corporation); Alison, M. R.[Tissue-based stem cells: ABC transporter proteins take center stage. JPathol. 2003 200(5): 547-50], Cai, J. et al., [Identifying and trackingneural stem cells. Blood Cells Mol Dis. 2003 31(1): 18-27] and Collins,A. T. et al., [Identification and isolation of human prostate epithelialstem cells based on alpha(2)beta(1)-integrin expression. J Cell Sci.2001; 114(Pt 21): 3865-72].

Briefly, in order to isolate adult stem cells of a fetal or a post nataltissue which includes dense matrix associated or connective proteins(such as elastin and collagen), the tissue can be first minced using ascalpel or a razor blade to small pieces (e.g., tissue chunks of 2-5mm), following which the tissue is subjected to an enzymatic digestionusing an enzyme such as dispase, collagenase (e.g., type I, II, III orIV), trypsin, natural protease, elastase, papain. Digestion enzymes canbe obtained from various suppliers such as Sigma (St Louis, Mo., USA)and Worthington. It will be appreciated that selection of the type ofdigestion enzyme for dissociating the biological sample containing theadult stem cells is depended on the composition of the proteins in thetissue and those of skilled in the art are capable of selecting thesuitable enzyme for each type of tissue. For example, for the digestionof tissues such as a placental tissue, chorionic villi sample (CVS),amniotic membrane, foreskin, bone, adipose tissue (fat) and looseconnective tissue which include collagen, type II collagenase may beused. On the other hand, for the digestion of a tissue which is rich inelastin such as the Wharton's jelly of the umbilical cord, elastase maybe used.

For example, a biological sample containing an adult tissue (e.g., anadipose tissue, CVS, placental tissue, amniotic membrane, cord,foreskin, bone, and loose connective tissue) can be dissociated usingtype II Collagenase at a concentration between 0.01-0.5%, preferably0.04-0.2%, most preferably 0.1%, at temperatures between 25-50° C.,preferably between 33-40° C., more preferably, at 37° C., for incubationperiods which may be effected from 30 minutes to 24 hours. It will beappreciated that the time of incubation with the digestion enzymedepends on the type of tissue. For example, while for adipose tissue,1-3 hours of digestion are sufficient, for bone tissue, completedigestion may require 18-24 hours or even more. Similarly, for digestionof CVS, placental tissue, amniotic membrane, the sample is incubatedwith the digestion enzyme, for digestion of, the sample is incubated for1-10 hours with the digestion enzyme, for digestion of foreskin, thesample is incubated over night with the digestion enzyme, for digestionof loose connective tissue, the sample is incubated over night with thedigestion enzyme. It will be appreciated that to increase digestionefficiency, the incubation with the digestion enzyme is preferablyperformed under agitation (e.g., by 40 RPM). It should be noted thatmeasures are taken in order to dissociate the large cell aggregates tosmall cell clumps or preferably to single cells without hampering thecell structure and viability. It will be appreciated that the morphologyof the dissociated adult stem cells can be monitored using an invertedmicroscope and the viability of the cells can be measured by subjectinga sample of the cells to the Live/Dead viability assay (MolecularProbes, Molecular Probes, Inc., Eugene, Oreg., USA).

Preferably, following enzymatic digestion, the biological samples aresubjected to differential centrifugation as is further describedhereinunder.

Alternatively, isolating adult stem cells from tissue samples which aredevoid of or have non-significant amounts of collagen, such as cordblood and bone marrow, can be performed by directly subjecting thesample to a differential centrifugation according to known techniques asdescribed elsewhere [Halvorsen, et al, Metabolism 2001, 50:407-413;Hauner, et al, J Clin Invest 1989, 84:1663-1670; Rodbell, et al, J BiolChem 1966, 241:130-139].

Differential centrifugation can be performed in a culture medium or overa gradient such as Ficoll or Percoll. Cells are centrifuged at speeds ofbetween 100 to 3000×g, more preferably 200-1500×g, more preferably at500×g, for periods of between 1 minute to 1 hour, more preferably 2 to15 minutes, more preferably 5 minutes, at temperatures of between 4-50°C., preferably between 20-40° C., more preferably at 25° C. Thus, cells

Still alternatively, isolation of adult stem cells from amniocytes canbe performed by directly subjecting the amniotic fluid sample tocentrifugation which separates the cells from the amniotic fluid.

As mentioned, once isolated the adult stem cells are cultured in aculture medium comprising cortisol and ascorbic acid.

Culturing the adult stem cells according to this aspect of the presentinvention is effected by seeding the adult stem cells in a culturevessel (e.g., a tissue culture plate, flask, container or bottle) at acell density which promotes differentiation of the adult stem cells intoconnective tissue progenitor cells which are capable of proliferation inculture while maintaining their multipotent capacity. For example, asuitable cell density which can be used to generate the CTPs of thepresent invention may be 1×10⁵-1×10⁶ cells per cm², more preferably,5×10⁵-1×10⁶ cells per cm² (e.g., 5×10⁵ cells per cm²). Culturingconditions usually include incubation of the cells at physiologicaltemperatures in the range of 35-38° C. (preferably, 37° C.), underhumidity and in the presence of 5% CO₂.

The culture medium used by the method of this aspect of the presentinvention may be any culture medium capable of supporting the growth ofthe CTPs of the present invention while maintaining their proliferativeand multipotent capacities. Such a culture medium can be a water-basedmedium which includes a combination of substances such as salts,nutrients, minerals, vitamins, amino acids, nucleic acids, proteins suchas cytokines, growth factors and hormones, all of which are needed formaintaining CTP proliferation and differentiation levels. For example, aculture medium according to this aspect of the present invention can bea synthetic tissue culture medium such as alpha-MEM (BiologicalIndustries, Beit Haemek, Israel), Ko-DMEM (Gibco-Invitrogen Corporationproducts, Grand Island, N.Y., USA), DMEM/F12 (Biological Industries,Biet Haemek, Israel), supplemented with the necessary additives as isfurther described hereinunder. Preferably, all ingredients included inthe culture medium of the present invention are substantially pure, witha tissue culture grade.

As used herein the term “cortisol” refers to a synthetic or naturallyoccurring cortison (a glucocorticoid steroid hormone) which binds theglucocorticoid receptor and is capable of expanding CTPs and maintainingtheir differentiation state. It will be appreciated that the method ofthis aspect of the present invention can use a variety of cortisolderivatives or analogues which are capable of the biological activity(e.g., expanding CTPs and maintaining their differentiation state).Non-limiting examples of cortisol derivatives or analogues which can beused according to this aspect of the present include hydrocortisone,cortisone acetate, prednisone, prednisolone, methylprednisolone,dexamethasone (about 40 times more potent), betamethasone,triamcinolone, beclometasone, fludrocortisone acetate anddeoxycorticosterone acetate (DOCA). Preferably, the cortisol analogueused by the method of this aspect of the present invention isdexamethasone, which is known to have a potent activity (about 40 timesmore than hydrocortisone) mediated by the glucocorticoid receptor.

The dexamethasone which is included in the culture medium of this aspectof the present invention can be obtained from Sigma (St Louis, Mo., USA)and is provided at a concentration selected from the range of 10⁻⁵-10⁻⁸M, e.g., at least 10⁻⁷ M. For example, as described under “GeneralMaterials and Experimental Methods” of the Examples section whichfollows, the present inventors have used 10⁻⁷ M dexamethasone within theculture medium to obtain a highly proliferative, multipotent CTPs.

As used herein “ascorbic acid” refers to the synthetic or naturallyoccurring ascorbic acid (an organic acid with antioxidant properties),also known as vitamin C, which is suitable for culturing cells (e.g.,sterile preparation).

The ascorbic acid which is included in the culture medium of the methodof this aspect of the present invention can be obtained from Sigma (StLouis, Mo., USA) and is provided at a concentration of at least 20μg/ml, more preferably, at least 30 μg/ml, preferably, at aconcentration which is selected from the range of 20-500 μg/ml, morepreferably, at a concentration which is selected from the range of20-200 μg/ml, more preferably, at a concentration which is selected fromthe range of 30-100 μg/ml, e.g., 50 μg/ml. For example, as describedunder “General Materials and Experimental Methods” of the Examplessection which follows, the present inventors have used 50 μg/ml ascorbicacid within the culture medium to obtain a highly proliferative,multipotent CTPs.

As is shown in FIGS. 8 a-d and is described in Example 3 of the Examplessection which follows, the CTPs generated by the method of this aspectof the present invention are capable of forming mineralized matrix.Preferably, in order to increase the deposition of mineralized matrix(e.g., when differentiation into a bone tissue is desired), the culturemedium further includes inorganic phosphate such asbeta-glycerophosphate, which can be obtained from Sigma (St Louis, Mo.,USA). The inorganic phosphate included in the culture medium is providedat a concentration in the range of 0.01-1000 mM, preferably, at aconcentration of 1-50 mM, even more preferably, at a concentration of 10mM.

Preferably, the culture medium used according to the method of thisaspect of the present invention further comprises serum or serumreplacement (e.g., a defined preparation made of mainly synthetic orrecombinantly expressed proteins which replaces the serum in a culturemedium). Such sera can be derived from a human source (human serum) orfrom an animal source (e.g., bovine serum, horse serum) and can beobtained from a variety of tissue culture suppliers such as Hyclone(Utah, USA), Invitrogen (Grand island, N.Y., USA) or BiologicalIndustries (Biet Haemek, Israel). A preparation of serum replacement canbe obtained from Gibco-Invitrogen Corporation (Grand Island, N.Y. USA).

The serum or serum replacement used by the method of this aspect of thepresent invention is provided at a concentration range of 1% to 40%,more preferably, 5% to 35%, more preferably, 10% to 30%, more preferably10% to 20%, e.g., 15%.

Preferably, culturing according to this aspect of the present inventionis effected under xeno-free culturing conditions, i.e., under conditionswhich are devoid of animal contaminants (e.g., animal pathogens such asretroviruses). Thus, the serum which is included in the culture mediumof the present invention is preferably human serum or serum replacement,and culture medium additives (e.g., the growth factors, minerals,vitamins or hormones such as dexamethasone and ascorbic acid) arepreferably synthetic or recombinantly expressed highly pure additives.

It will be appreciated that the CTPs of the present invention can growin the above-described culture medium by either directly attaching tothe wall of the culture vessel (e.g., the bottom of a culture flask) orby attaching to feeder cells which are attached to the wall of theculture vessel. Feeder cells are cells such as stromal cells, fibroblastcells (e.g., foreskin fibroblasts, human embryonic fibroblasts) or humanfallopian epithelial cells, which secrete growth factors and/ornutrients and support the growth of the cells-of-interest (e.g., stem orprogenitor cells) cultured therewith. However, it will be appreciatedthat culturing conditions which rely on feeder cells are much morecomplicated since they require the separation of the cells-of-interestfrom the feeder cells at each passaging step and thus may result inmixed populations of cells.

Preferably, culturing according to the method of this aspect of thepresent invention is effected under feeder-free culturing conditions. Asused herein the phrase “feeder-free culturing conditions” refers toculturing conditions which are devoid of feeder cells. For example, asshown in the Examples section which follows, the CTPs of the presentinvention (which are cultured under feeder-free culturing conditions)are completely devoid of feeder cells and thus represent a purepopulation of CTPs.

Preferably, the method of this aspect of the present invention furthercomprising passaging the connective tissue progenitor cells in apresence of the culture medium which comprises cortisol (e.g.,dexamethasone) and ascorbic acid, wherein a first passage of thepassaging is effected no more than 10 days following initial culturingof the adult stem cells in the culture medium.

The term “passaging” as used herein refers to splitting the cells sothat the cells can grow beyond confluency. The term “confluency” as usedherein refers to a density of adherent cells which when attached to awall of a culture vessel cover 100% of its area. Preferably, in order toprevent the differentiation of the CTPs of the present invention toterminally differentiated cells (e.g., osteoblasts) or to cells withlimited differentiation potential (e.g., precursors which are restrictedto a specific cell lineage), the first passaging occurs when the cellsin the culture reach subconfluency, i.e., when the cells cover about70-90% of the culture vessel wall, more preferably, when the cells coverabout 80-90% of the culture vessel wall. Preferably, when culturedaccording to the teachings of the present invention subconfluency of thecells in the culture occurs no more than 10 days of the initial seedingof the cells. Preferably, the cells are serially passaged after no morethan 9 days in culture, more preferably, after no more than 8 days inculture, more preferably, after no more than 7 days in culture, morepreferably after about 4-6 days in culture.

Passaging according to this aspect of the present invention can beperformed by dissociating cells from the wall of the culture vesselusing e.g., type IV collagenase (at a concentration of 0.1% for 20-60minutes) followed by trypsinization (using 0.25% trypsin for 2-5minutes), counting the single cells and splitting the cells to 2-5,preferably to 4 tissue culture vessels (i.e., a splitting ratio of 1:4)in order to preserve the same cell density of their initial seeding(e.g., 5×10⁵-1×10⁶ cells per cm²). Preferably, the cell culture issubjected to culture passaging every 3-6 days, preferably, culturepassaging occurs every 2-5 days, e.g., every 4 days.

Preferably, passaging according to the method of this aspect of thepresent invention is performed for at least 20 times, more preferably,at least 25 times, more preferably, at least 30 passages, whilesplitting the cells using a splitting ratio of 1:3, more preferably, asplitting ratio of 1:4 (a splitting ratio of 1:4). As described in theExamples section which follows, the CTPs of the present invention wereserially passaged every 4 days for at least 25 passages.

Preferably, passaging according to the method of this aspect of thepresent invention is effected under xeno-free and/or feeder-freeconditions.

In addition, the method of this aspect of the present inventionpreferably comprises a step of isolating the CTPs (i.e., separating theCTPs from the culture medium). Isolating the cells can be effected atany time while in culture, e.g., prior to the first culture passaging,following the first culture passaging, or following any other culturepassaging. Thus, the CTPs can be isolated by filtration (e.g., using amembrane which prevents the passage of CTPs and enables passage of themedium and/or by centrifugation of the cells (e.g., using a conical tubecentrifugation at 1500 RPM).

Preferably, the method of this aspect of the present invention furthercomprises expanding the CTPs. As used herein the phrase “expanding”refers to increasing the number of the connective tissue progenitorcells over the culturing period without hampering their differentiationcapacity.

Preferably, CTPs generated and isolated according to the method of thisaspect of the present invention are highly proliferative cells. Forexample, adult stem cells-derived CTPs exhibited a tripling time (i.e.,the hours between passages in which the cell number was triplicated) of80-100 hours during passages 1-18 and of 120-140 hours during passages19-25 (data not shown).

Preferably, expanding of the connective tissue progenitor cells in theculture medium which comprises cortisol (e.g., dexamethasone) andascorbic acid is effected such that at least 3.5×10⁹ connective tissueprogenitor cells are obtained from a single cell of the adult stem cellsfollowing about 20 passages.

As is shown in FIGS. 1 a-g and is described in Example 2 of the Examplessection which follows, FACS analysis revealed that the CTPs isolatedaccording to the method of this aspect of the present invention expresson their cell surface CD105 (a marker of endothelial cells, also knownas Endoglin, GenBank Accession No. NP_(—)000109.1), CD166 (activatedleukocyte cell adhesion molecule, also known as ALCAM, GenBank AccessionNo. NP_(—)001618.2), CD44 (e.g., GenBank Accession No. NP_(—)000601.3,NP_(—)001001389.1, NP_(—)001001390.1, NP_(—)001001391.1,NP_(—)001001392.2), CD29 (ITGB1; e.g., GenBank Accession No.NP_(—)002202.2, NP_(—)391987.1, NP_(—)391989.1, NP_(—)391988.1,NP_(—)596867.1, NP_(—)389647.1) and HLA-ABC. On the other hand, the CTPsof the present invention do not express CD45 (PTPRC; e.g., GenBankAccession No. NP_(—)002829.2, NP_(—)563579.1, NP_(—)563578.1,NP_(—)563580.1) and HLA-DR, thus resembling the expression profile ofother mesenchymal stem cells like cells.

As used herein the phrases “expressing” or “not expressing” refer tocells having a positive (+) or negative (−) expression profile,respectively, of a certain marker (e.g., gene or gene product).

Thus, the present invention provides an isolated cell preparation ofadult stem cells—derived CTPs. Preferably, the connective tissueprogenitor cells of the isolated cell preparation are capable of beingmaintained in a proliferative, non terminally differentiated state forat least 20 passages in culture.

The phrase “non terminally differentiated state” as used herein refersto cells which are capable of differentiating to at least two celllineages of the connective tissue even following 20 passages in culture.For example, as is shown in FIGS. 2 a-u and is described in Example 2 ofthe Examples section which follows, adult stem cells-derived CTPs fromvarious passages (e.g., from passages 1-25) express markers ofosteogenic lineage, the chondrogenic lineage, cartilage cells, tendoncells, ligament cells and ECM-forming cells. Moreover, as described inthe Examples section which follows, when cultured in vitro in thepresence of inorganic phosphate, the adult stem cells—derived CTPsformed bone nodules (bone tissue) containing calcium-phosphate deposits,the major component of bone minerals. In addition, when induced todifferentiate into the chondrogenic lineage (using the “intact layer”method), adult stem cells—derived CTPs formed a fibrous ECM (FIGS. 9a-b, Examples 4 of the Examples section which follows).

Thus, while further reducing the present invention to practice, thepresent inventors have uncovered that the CTPs of the present inventioncan be used to generate a connective tissue.

Preferably, the connective tissue which can be formed from the CTPs ofthe present invention is a bone tissue (e.g., osseous tissue), aconnective tissue (e.g., loose connective tissue), an extracellularmatrix (ECM), a tendon tissue, a ligament tissue and a cartilage tissue.

For example, to form a bone tissue, the isolated cell preparation of theCTPs of the present invention are cultured in a medium containingdexamethasone, ascorbic acid and inorganic phosphate (e.g., α-MEMsupplemented with 15% serum, 10⁻⁷ M dexamethasone, 50 μg/ml ascorbicacid and 10 mM (β-glycerphosphate) and let to become over-confluent(i.e., they occupy more than 100% of the area of the culture vessel walland thus form multilayers) for a period of at least 10 days beforemineralization appears. The culture medium is occasionally replacedevery 4-8 days without culture passaging. The presence of bone tissuecan be determined by RNA-based methods (e.g., RT-PCR, in situ RNAhybridization and cDNA microarray) or protein-based methods (e.g.,immunological staining) for bone specific markers such as core bindingfactor alpha 1 (CBFA1), SOX9, type I collagen (Col-I), osteonectin,osteopontin, parathyroid hormone receptor 1 (PTHR1) and alkalinephosphatase. In addition, the ultrastructural and chemical data of bonetissue can be determined using, for example, energy dispersivespectroscopy (EDS), scanning electron microscopy (SEM) and/or confocalRaman spectroscopy (CRS) analyses essentially as described elsewhere[Ubelaker D H, et al., J Forensic Sci. 2002 September; 47(5):940-3; vanApeldoorn A A, et al., J R Soc Interface. 2005 Mar. 22; 2(2):39-45].

For the formation of connective tissue, the isolated cell preparation ofthe CTPs of the present invention are preferably seeded on a suitable 3Denvironment (e.g., a scaffold such as an electrospun, PCL/PLA nanofiberscaffold) that would support their growth and organization into acomplex connective tissue which produces ECM. Briefly, 5×10⁵ CTPsresuspended in 10 μl of CTP medium (α-MEM supplemented with 15% serum,10⁻⁷ M dexamethasone, 50 μg/ml ascorbic acid) are seeded on a scaffoldand grown in the presence of the CTP medium for about one month. Thepresence of connective tissue and ECM can be determined by RNA orprotein-based methods for markers such as type III (Col-III) and typeXII (Col-XII) collagens, decorin, biglycan, elastin, fibronectin, andtenascin-C, as well as using scanning electron microscopy (SEM)analysis.

For the formation of extracellular matrix (ECM), the isolated cellpreparation of CTPs are plated at high densities in tissue cultureplates in the presence of a culture medium comprising ascorbic acid andpreferably also dexamethasone. After about 4 weeks in culture (otherperiods of time are possible too) sheet-like tissue is formed. Thesheet-like tissue can be further subject to freeze-drying and/or cellremoval as shown in FIGS. 15-18 and is described in Example 10 of theExamples section which follows. Thus, the ECM can be intact matrix(which can be freeze dry, and/or subjected to acellularization forfurther tissue engineering applications, tissue replacement, tissuerepair and the like, and/or it can be a soluble ECM which can be used asan injectable material for tissue engineering applications, tissuereplacement, tissue repair and the like.

For the formation of ligament-forming cells, the isolated cellpreparation of the CTPs of the present invention are cultured for atleast one week in the presence of medium containing dexamethason andascorbic acid (e.g., α-MEM supplemented with 15% serum, 10⁻⁷ Mdexamethasone, 50 μg/ml ascorbic acid). The presence of ligament-formingcells can be detected by RNA or protein-based methods for markers suchas scleraxis and type I Collagen.

For the formation of a cartilage tissue, two main methods can beemployed: the “intact layer” method (removal of sub-confluent CTPcultures without pre-collagenase treatment and culturing the intactlayer in a CTP medium, essentially as described in the Examples section)and the “pellet culture” method (spherical pellets which are cultured inmedium supplemented with serum, dexamethasone, ascorbate-2-phosphate andTGF-β3; essentially as described in the Example section). The presenceof cartilage tissue can be detected by RNA or protein-based methods formarkers such as chondroitin sulfate proteoglycan 4 (CS4), type Xcollagen, and cartilage oligomeric matrix protein (COMP).

As described in the Examples section which follows, the presentinventors have devised a long-term, high-density culture technique forthe formation of a tendon tissue. Thus, the CTPs of the presentinvention were cultured in vitro at a cell density of 5-10×10⁶ cells/cm²in the presence of a culture medium containing alpha-MEM supplementedwith 15% FBS, 50 μg/ml ascorbic acid and 10⁻⁷ M dexamethasone. Cultureswere kept for long periods of up to 4 months in culture, with no furthersplitting. As is shown in FIGS. 5 a-f and is described in Example 6 ofthe Examples section which follows, tendon tissues formed in vitroincluded organized, parallel-aligned cells that express type I collagenand exhibit high matrix-to-cell ratio. Moreover, such tendon tissuesexhibited mechanical properties which are similar to the native tendontissues (Example 6).

Thus, according to yet another aspect of the present invention there isprovided a method of generating a tendon tissue in vitro.

The method is effected by culturing the CTPs of the present invention ina culture medium which comprises ascorbic acid and/or dexamethason underculture conditions devoid of a carrier, thereby generating the tendontissue.

The term “carrier” refers to any scaffold, bead, polymer or matrix whichsupports the attachment of cells thereto. The phrase “devoid of acarrier” as used herein refers to any culture conditions which enablethe attachment of the CTPs directly to the culture vessel wall and notto a carrier (e.g., a scaffold). This is in sharp contrast to all knownmethods of generating tendon tissues in vitro, which are based onseeding cells-of-interest (e.g., non-genetically modified cells) onto ascaffold or a polymer suitable for tissue formation.

Preferably, the culture medium used for generating the tendon tissuecomprises ascorbic acid (e.g., between 1-500 μg ml, preferably, about 50μg/ml) and dexamethasone (e.g., between 10⁻⁵-10⁻⁸ M, preferably, about10⁻⁷ M).

Preferably, culturing according to the method of generating a tendontissue of this aspect of the present invention is effected without cellpassaging for culturing periods which vary between a few weeks toseveral months (e.g., between 4 weeks to 6 months). Preferably, theculture medium is occasionally replaced, e.g., every 2-7 days (e.g.,every 4 days). It will be appreciated that the process of tendon tissueformation begins as a single cell attaching to the culture plate sidewall, forming fibrous matrix with a tendon-like shape.

The presence of a functional tendon tissue can be determined usinghistological staining, immunological assays (e.g., using an anti-type Icollagen antibody), SEM analyses, electron microscopy, and mechanicalevaluation using the stress strain test, essentially as described in theExamples section which follows.

It will be appreciated that for engineering of a particulartissue-of-interest (e.g., a tendon or a ligament), the culturing periodof the cells may vary (e.g., become shorter) such that following theformation of a sheet-like tissue (e.g., following 4-5 weeks), thesheet-like tissue is removed from the culture vessel (using e.g., a cellscraper) and further rolled or folded to form the engineeredtissue-of-interest (e.g., tendon, ligament). It will be appreciated thatdue to the culturing conditions employed according to this aspect of thepresent invention, which result in a tissue with high matrix to cellsratio, removal of the sheet-like tissue can be easily performed whilepreserving the intact tissue structure.

For example, as is illustrated in FIGS. 13 a-e and is described inExample 9 of the Examples section which follows, following 4-5 weeks inthe high density culture the formed sheet-like tissue was removed androlled to form a rounded cylinder. The resulting tendon tissues werecylinder-shaped constructs, with typical ultrastructure characteristicsand biomechanical properties of early tendons.

It will be appreciate that tissues which are formed in vitro from theisolated CTPs of the present invention (engineered tissues) can befurther implanted in a subject in need thereof (e.g., a subject in needof a CTP-derived tissue formation, regeneration and/or repair) usingtechniques known in the art (e.g., using a surgical tool such as ascalpel, spoon, spatula, suture device, or other surgical device) tothereby regenerate, replace and/or repair the tissue-of-interest.

For example, as is shown in FIGS. 14 a-b and is described in Example 9of the Examples section which follows, the present inventors werecapable of repairing a critical Achilles-tendon injury in mice. Thus,the implanted tendon grafts were remodeled and exhibited excellentbiomechanical properties. Moreover, the implanted tendon grafts werefunctional in vivo as evidenced by the increased ankle extensionfollowing transplantation of a tendon graft instead of the injuredAchilles tendon.

While further reducing the present invention to practice, the presentinventors have uncovered that the connective tissue progenitor cellsobtained according to the method of this aspect of the present inventioncan be used for in vivo cell-based therapy.

As described in Example 7 of the Examples section which follows, whenthe adult tissue-derived CTPs of the present invention were implantedunderneath the kidney capsule of SCID-beige mice a localizedsub-capsular formation of loose connective tissue was observed. Inaddition, as described in Example 8 of the Examples section whichfollows, when the adult tissue-derived CTPs were subcutaneouslytransplanted into cd1 nude mice, ectopic new bone and cartilage tissueswere formed. The ectopic tissues were well vascularized andbiocompatible (FIGS. 11 a-c) and included radio-opaque bone tissue (FIG.4). Further histological analyses of the ectopic tissues confirmed theformation of new bone (FIGS. 12 a-b) and hypertrophic cartilage (notshown) tissues.

Thus, according to yet another aspect of the present invention there isprovided a method of in vivo forming or generating a connective tissue.The method is effected implanting in a subject in need therefore theconnective tissue progenitor cells of the present invention; thereby invivo forming the connective tissue.

The phrase “in vivo” refers to forming a tissue within a living organismsuch as a plant or an animal, preferably in mammals, preferably, inhuman subjects.

The phrase “a subject in need thereof” as used herein refers to amammal, preferably a human being at any age who is in need of aconnective tissue such as for tissue construction, repair, regenerationor replacement. For example, such a subject can suffer from a diseased,degenerated, injured or broken tissue or may be missing a particulartissue.

Preferably, the connective tissue which can be formed in vivo accordingto the method of this aspect of the present invention include bonetissue, cartilage tissue and loose connective tissue.

Implanting the CTPs in the subject can be performed using methods knownin the art such as by administering or injecting the CTPs with a syringeneedle, catheter or cannula. The cells are preferably administered nearor at the site-of-interest (e.g., bone, cartilage, connective tissue)within the subject (e.g., at the site of the damaged or injured tissue)and thus can be used to repair bone fracture, diseased or damagedcartilage, bone or connective tissue. It will be appreciated that insome cases alternative sites may be used. In addition, it will beappreciated that the cells can be administered as an isolated cellpreparation (cell therapy) or can be first seeded on a scaffold and thenadministered to the subject (engineered tissue therapy).

The present invention further contemplates monitoring the formation,regeneration or repair of the connective tissue following implantation.For example, the formation of new bone tissue can be monitored by X-rayand/or CT analysis; the formation of a new tendon tissue can bemonitored by MRI; the formation of a new connective tissue can bemonitored by MRI. Alternatively, the formation of new tissue can beevaluated by physiological assays such as extension of a tendon,strength of a bone and the like.

It will be appreciated that the CTPs of the present invention can bederived from either autologous sources such as self bone marrow cells,self cord blood cells, self foreskin cell, self adipose tissue, or fromallogeneic sources such as bone marrow, bone, umbilical cord, cordblood, connective tissue or other cells derived from non-autologoussources. Since non-autologous cells may induce an immune reaction whenadministered to the body several approaches have been developed toreduce the likelihood of rejection of non-autologous cells. Theseinclude either suppressing the recipient immune system or encapsulatingthe non-autologous cells or tissues in immunoisolating, semipermeablemembranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu M Z, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesThechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, the quality control, mechanical stability,diffusion properties, and in vitro activities of encapsulated cellsimproved when the capsule size was reduced from 1 mm to 400 μm (CanapleL. et al., Improving cell encapsulation through size control. J BiomaterSci Polym Ed. 2002; 13: 783-96). Moreover, nanoporous biocapsules withwell-controlled pore size as small as 7 nm, tailored surface chemistriesand precise microarchitectures were found to successfully immunoisolatemicroenvironments for cells (Williams D. Small is beautiful:microparticle and nanoparticle technology in medical devices. Med DeviceTechnol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology forpancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

The isolated cell preparation of CTPs of the present invention may alsoform part of a pharmaceutical composition.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the isolated cellpreparation of connective tissue progenitor cells of the presentinvention accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Thus, the teachings of the present invention can be used for varioustherapeutic applications such as tendon and ligament repair, boneregeneration, cartilage regeneration, tissue augmentation, tissuereconstruction (mediated or assisted by the CTPs of the presentinvention) such as deep wound healing, burn wound dressing and skinregeneration, skin fillers, orthodontic procedures, sling procedures andfascia reconstruction.

The present invention further contemplates an article-of-manufacturewhich comprises packaging material and a composition comprising theisolated cell preparation of CTPs of the present invention along withinstructions for use in cell based therapy, tissue repair,reconstruction, regeneration and/or replacement.

The present invention further contemplates an article-of-manufacturewhich comprising packaging material and a tissue graft generated invitro from the CTPs of the present invention along with instructions forusing the tissue graft for tissue repair, regeneration and/orreplacement.

The present invention further contemplates the use of the CTPs of thepresent invention for the preparation of intact ECM (non-conditioned orconditioned with certain cells such as specialized cells, e.g.,cardiomyocytes, keratinocytes and the like) for specialized tissueregeneration/regeneration as well as purified ECM components for tissueregeneration, anti-aging medicine related applications.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

General Materials and Experimental Methods

Connective Tissue Progenitors (CTPs) Derivation and Propagation FromAdult Stem Cells—

Adult stem cells derived CTPs were generated from fetal connectivetissue including bone, muscle, cartilage and loose connective tissue,from termination of pregnancies from 9-16 weeks of gestation. Inaddition, adult stem cells derived CTPs were generated from adult tissueincluding foreskin (from a neonatal individual) and fat tissue (from20-60 year old individuals) obtained via liposuction. Briefly, adiposetissue was washed with PBS solution and then subjected to 0.1-0.2% typeII collagenase digestion for 1-4 hours. The stromal vascular fractionwas then separated from the adipose cells by centrifugation and furtherpurified, using plastic adherence, to remove contaminating mononuclearcell fractions. Next cells were introduced with CTP medium. For foreskinCTPs, foreskin tissue was washed with PBS solution and the tissue wascut into 2-4 mm pieces, and then subjected to 0.1-0.2% type IIcollagenase digestion for overnight. Next cells were introduced with CTPmedium. For fetal tissue CTP, fetal tissue was washed with PBS solutionand the tissue was cut into 2-4 mm pieces, and then subjected to0.1-0.2% type II collagenase digestion for overnight. Alternatively, forfetal CTPs, the fetal tissue was cut into 2-4 mm pieces, and thendirectly seeded on tissue culture plates and introduced with CTP medium.

Osteogenic Differentiation and Matrix Formation—

For induction of mineralization, CTPs were grown with CTP mediumsupplemented with 10 mM beta-glycerophosphate (inorganic phosphate), andlet to become over-confluent for period of at least 10 days beforemineralization appears.

Chondrogenic Differentiation

Method I: The “Intact Layer” Method, Formerly Called “Spontaneous”—

Sub-confluent CTP cultures (from any passage, e.g., 1-13) were removedfrom the culture plates (without pre-collagenase treatment) as an intactlayer, were placed in suspension and were fed with the CTP mediumdescribed hereinabove. This method generates typical cartilagemorphology.

Method 2: The Pellet Culture System, Formally Called “Directed”—

Sub-confluent CTP cultures (from any passage) were re-suspended at adensity of 2—10⁵ cells/ml (the density can vary), dispensed into 15-mlconical tubes and centrifuged for 5 minutes at 1,200 rpm to formspherical pellets. The pellets were further cultured in mediumcontaining 1% serum in addition to high-glucose Dulbecco's modifiedEagle's medium supplemented with 10⁻⁷ M dexamethasone, 50 μg/mlascorbate-2-phosphate, 40 μg/ml L-proline, 100 μg/ml sodium pyruvate, 50mg/ml ITS+Premix (Collaborative Biomedical: 6.25 μg/ml insulin, 6.25μg/ml transferrin, 6.25 ng/ml selenious acid, 1.25 mg/ml bovine serumalbumin, and 5.35 mg/ml linoleic acid) and 10 ng/ml TGF-β3. This methodinduced up-regulation of specific cartilage matrix genes such as Col-X,COMP and CS4.

RT-PCR Analysis—

Total RNA was isolated using the TRIzol Reagent (Invitrogen, Carlsbad,Calif. USA) according to the manufacturer's instructions. cDNA wasreverse transcribed from 1 μg total RNA with M-MLV Reverse Transcriptase(Promega, Madison, Wis. USA). PCR conditions were optimized for each setof primers and the number of PCR cycles was confirmed to be in thelinear range of amplification. The amplified products were separated on2% agarose gel stained with ethidium bromide and identified by size. Theidentity of each product was confirmed by restriction enzyme digestion.Samples not treated with reverse transcriptase and no-template sampleswere used as controls. Table 1, hereinbelow, summarizes the primersequences (along with their SEQ ID NOs.), annealing temperatures, cyclenumbers used for RT-PCR, the restriction enzyme used to verify theidentity of RT-PCR products (along with the expected size of thedigested product).

TABLE 1 RT-PCR primers and conditions Gene RT-PCR (GenBankForward (F) and Reverse (R) Restr. product Accession No.)primers (SEQ ID NO: ) 5′→3′ Cyc. Ann. Enzyme size CBFA1 CBFA1 F: 40 62°C. SacI 283 bp (NM_004348) CCGCACGACAACCGCACCAT (SEQ ID 30 sec 215 bpNO: 1) CBFA1 R: CGCTCCGGCCCACAAATCTC (SEQ ID NO: 2) SOX9Sox9 F: ATCTGAAGAAGGAGAGCGAG 35 58° C. EcoRII 264 bp NM 000346(SEQ ID NO: 3) 30 sec Sox9 R: TCAGAAGTCTCCAGAGCTTG (SEQ ID NO: 4) COL1hCOL1 F: 35 64° C. NcoI 476 bp NM 000089.3 GCACACAATGGATTGCAAGG (SEQ ID30 sec 39 bp NO: 5) BclI hCOL1 R: TAACCACTGCTCCACTCTGG 45 bp(SEQ ID NO: 6) ONEC Onec F: GCAGCAATGACAACAAGACC 35 58° C. SphI 277 bpNM 003118 (SEQ ID NO: 7) 30 sec 166 bp Onec R: CTTCTCATTCTCATGGATCTTC(SEQ ID NO: 8) OPN hOPN F: 30 55° C. EcoRII 330 bp NM_000582.21CTAGGCATCACCTGTGCCATACC (SEQ 45 sec 226 bp ID NO: 9) hOPN R:CAGTGACCAGTTCATCAGATTCATC (SEQ ID NO: 10) ALPALP F: TGGAGCTTCAGAAGCTCAAC 35 62° C. BstXI 435 bp NM 000478(SEQ ID NO: 11) 30 sec 120 bp ALP R: ATCTCGTTGTCTGAGTAGTACCAGTCC(SEQ ID NO: 12) Hpth/r1 hPTH/R F: CACAGCCTCATCTTCATGG 35 60° C. SacI417 bp NM 000316 (SEQ ID NO: 13) 45 sec 148 bp hPTH/R1 R:GCATCTCATAGTGCATCTGG (SEQ ID NO: 14) COL9 α2Col9/2 F: TGGTTTAACTGGAGCCAAGG 35 60° C. SphI 520 bp (17/11)(SEQ ID NO: 15) 30 sec 330 bp NM_001852.31Col9/2 R: GCCCACCATGAATTTATATC (SEQ ID NO: 16) COL10 COL10 F: 40 60° C.XhoI 468 bp NM 000493 CCCTTTTTGCTGCTAGTATCC (SEQ ID 30 sec 194 bpNO: 17) COL10 R: CTGTTGTCCAGGTTTTCCTGGCAC (SEQ ID NO: 18) COMPCOMP F: CAGGACGACTTTGATGCAGA 35 57.5° C. BstXI 314 bp (NM_000095)(SEQ ID NO: 19) 30 sec 141 bp COMP R: AAGCTGGAGCTGTCCTGGTA(SEQ ID NO: 20) AGGRECAN AGN F: ATCCGAGACACCAACGAGAC 35 60° C. SphI477 bp NM 001135 (SEQ ID NO: 21) 30 sec 290 bpAGN R: GGCTTCACCCTCACTGATGT (SEQ ID NO: 22) CS4CS-4S F: CCCCCATCCTCACTACAAAC 40 60° C. PstI 242 bp NM 001897(SEQ ID NO: 23) 30 sec 34-38 CS-4S R: ATCCAGGGTTCCTCTGTGTG bp(SEQ ID NO: 24) SCLER Scler F: TGCAAGCTTCCCTTTTCAGT 40 60° C. HgaI455 bp BK000280.11 (SEQ ID NO: 25) 30 sec 292 bpScler R: CTGCACAGCCGAAATTGTAA (SEQ ID NO: 26) COL3Col3 F: CCTCCAACTGCTCCTACTCG 35 60° C. BglI 439 bp NM_000090.21(SEQ ID NO: 27) 30 sec 266 bp Col3 R: CGGGTCTACCTGATTCTCCA(SEQ ID NO: 28) collagen, type Col-XII F: GTGCCTGGACTGATTTGGTT 35 60° C.ScaI  464 bp XII (SEQ ID NO: 29) 30 sec 185 bp NM_004370 Col-XII R: StyITGTGGAGGCAATTTGTTTGA (SEQ ID 414 bp NO: 30) DECORDecor F: TGAAGAACCTTCACGCATTG 35 60° C. SacI 481 bp NM_001920.31(SEQ ID NO: 31) 30 sec 255 bp Decor R: GAGCCATTGTCAACAGCAGA(SEQ ID NO: 32) BIGLY Bigly F: TGCAGAACAACGACATCTCC 35 60° C. BstXI319 bp NM_001711.31 (SEQ ID NO: 33) 30 sec 192 bpBigly R: CCAGGTTCAAAGCCACTGTT (SEQ ID NO: 34) ELASTElast F: GCTATGGACTGCCCTACACC 40 60° C. BglI 371 bp NM_000501.11(SEQ ID NO: 35) 30 sec 288 + Elast R: AGCTCCTGGGACACCAACTA 329 bp(SEQ ID NO: 36) FIBRO Fibro F: GGAGTCAGCTGCCAAGAGAC 35 60° C. XhoI482 bp NM 212482 (SEQ ID NO: 37) 30 sec 215 bpFibro R: ACACACGTGCACCTCATCAT (SEQ ID NO: 38) TEN CTen F: CGTGGAGTACCTTGTCAGCA 35 60° C. XhoI 438 bp NM 002160(SEQ ID NO: 39) 30 sec 252 bp Ten R: AGGTAACCGGTGACTGATGC(SEQ ID NO: 40) Primers used to amplify RT-PCR products of the notedgenes (are referred to using GenBank Accession Nos.) are provided alongwith the PCR annealing (Ann.) conditions, number of PCR cycles (Cyc.),the size of PCR products and the restriction enzyme (Restr. Enzyme) anddigestion product used to verify the identity of the RT-PCR product.

Microarray Analysis—

For cDNA microarray analysis, isolated total RNA from undifferentiatedhESCs (H9.2), fetal CTPs (passage 1) and hESC-derived CTPs grown in thepresence of the CTP medium (passages 1 and 9) were reverse transcribedwith M-MLV Reverse Transcriptase (Promega, Madison, Wis. USA) usingBiotin-16-dUTP (Roche, Mannheim, Germany). The array membranes (GEArrayQ Series Human Osteogenesis Gene Array HS-026, SuperArray BioscienceCorp., Frederic, Md. USA) were pre-hybridized for 2 hours withheat-denatured salmon sperm DNA (Invitrogen) at a final concentration of100 μg/ml according to the manufacturer's instructions. The membraneswere hybridized overnight with Biotin-16-dUTP-labeled cDNA probes, andthen washed twice for 15 minutes each in a solution of 2×SSC, 1% SDSfollowed by two washes of 15 minutes each in a solution of 0.1×SSC, 0.5%SDS. All steps were performed at 60° C. with continuous agitation.Chemiluminescent detection was performed using the ChemiluminescentDetection Kit (SuperArray Bioscience Corp., Frederic, Md. USA) accordingto the manufacturer's instructions. The membranes were blocked for 40minutes with a GEAblocking solution Q. Binding of alkalinephosphatase-conjugated streptavidin was performed by incubating themembranes for 10 minutes in a binding buffer. The membranes were thenwashed four times, for 5 minutes each, with 1× Buffer F and rinsed twicewith Buffer G, followed by incubation of 2-5 minutes with CDP-starchemiluminescent substrate. All steps were performed at room temperaturewith continuous agitation. The signal was detected using X-ray film.

Karyotype Analysis—

For karyotype analyses, standard G banding was performed on adult stemcells derived-CTPs (from fetal tissue, CTPs were from passage 8-10, fromfat tissue, CTPs were from passage 14) essentially as describedelsewhere (Amit, M. et al, 2000).

Electron Microscopy—

For transmission electron microscopy (TEM) cells were fixed in 3%glutaraldehyde in 0.1 M sodium cacodylate buffer pH=7.4, post-fixed with1% OsO₄ and in 2% uranyl acetate, gradually dehydrated in ethanol seriesand embedded in Epon 812. Ultrathin sections (70 to 90 nm) cut on anultramicrotome (Leica UCT) were mounted on grids, stained withlead-citrate, and then examined with a Tecnai 12 transmission electronmicroscope under 120 kV. Pictures were digitized with CCD Megaview IIIand analyzed with analySIS software (Soft Imaging System).

Scanning Electron Microscopy (SEM)—

For SEM, cells and cell-seeded scaffolds were fixed in 3% glutaraldehydein 0.1 M sodium cacodylate buffer pH=7.4, followed by gradualdehydration in ethanol and drying using hexamethyldisilazane (HMDS)(Sigma). Samples were sputter coated with carbon and viewed under LEOfield-emission scanning electron microscope for imaging and energydispersive spectroscopy (EDS) analysis.

Cytochemistry and Electron Microscopy—

For histological analyses, cells were fixed in 10% natural bufferedformalin, gradually dehydrated in ethanol and embedded in paraffin.Sections were stained with Hematoxylin and Eosin (H&E) for generalhistomorphology. Picro-sirius red (Gurr-BDH, England) was used for thedetection of matrix collagens, and Toluidine blue (Serva, Germany) wasused to detect matrix proteogylcans in chondrogenic cultures (Hyllested,J. L, et al 2005). Alizarin Red (Sigma) was used to detect calciumphosphate deposits on culture plates as evidence for bonemineralization. For immunofluorescence studies, cells were fixed in 4%paraformaldehyde in phosphate-buffered saline (PBS), and stained withthe following primary antibodies, diluted in primary antibody diluent(Dako): anti-human type I collagen (Chemicon, Intnl, Inc. Temecula,Calif., USA, cat. No. MAB3391), anti-human alkaline phosphatase (R&D,Systems Inc, Minneapolis, Minn., USA, Cat. No. MAB1448) and anti-humantype II collagen (Chemicon, Cat. No. MAB8887). DAPI was used for nuclearcounterstaining. Appropriate secondary antibodies were used forvisualization.

FACS Analysis—

Cells were removed from the culture dish with type IV collagenase(Wortington) (0.1%, 30-60 minutes at 37° C.) followed by Trypsin/EDTA(Sigma) for 5 minutes at 37° C. and re-suspended in a FACS buffer[Sigma, PBS supplemented with 2% fetal bovine serum (Gibco) andcontaining 0.05% NaN3]. Fc blocking was performed by addition of 4%Human serum (Sigma). Cells were probed for 30 minutes at roomtemperature with the specific monoclonal antibodies (Pharmingen;eBioscience; DAKO) or an appropriate isotype control antibodies(Pharmingen; eBioscience; DAKO) in FACS buffer. Cell were washed twicein FACS buffer, and analyzed using a FACSAria flow cytometer (BectonDickinson). Acquisition was set for 10,000 events per sample. Dead cellswere excluded from analysis by staining with 7AAD (eBioscience). Datawere analyzed with Diva software (Becton Dickinson). Experiments wereperformed in duplicates.

Scaffold Fabrication and Cell Seeding—

Electrospun nanofiber scaffolds were made of a 1:1 blend ofpolycaprolactone (PCL) and poly (lactic acids) (PLA) by a processpreviously described (Ma, Z. et al 2005). The average thickness of theprepared scaffold was 500 μm, fiber diameter ranged between 200-450 nm,with porosity of 85%. For preparation for cell seeding, scaffold mat wascut into 0.5×0.5 cm² squares, gas-sterilized with ethylene oxide,immersed in 5 M sodium hydroxide and washed in PBS to increase surfacehydrophilicity. For seeding the cells on scaffolds, subconfluent CTPcultures were collagenase-treated, trypsinized and counted. 5×10⁵ cellswere resuspended in 10 μl of CTP medium, seeded onto each scaffold andallowed to attach for 30 minutes before fresh medium was added. Cultureswere maintained for one month before harvesting for analysis. Plaincultures on plastic plates were used as control.

In-Vitro Engineering of Functional Tendons—

Tendons were spontaneously formed upon applying the new long-termhigh-density culture technique: subconfluent CTP cultures (passages2-21) were regularly split and counted before seeding at a high densityof 5-10×10⁶ cells/cm² on tissue culture plates in the presence of CTPmedium. Cultures were kept for long periods of up to 4 months inculture, with no further splitting.

Mechanical Testing of In Vitro Engineered (Formed) Tendon Grafts—

Engineered constructs were removed from their culture plates immediatelybefore testing. The construct diameter was measured at several positionsalong the length, using an optical microscope (Olympus BX 60 ×500magnification). An average diameter was calculated, using measuredvalues. Tensile testing was performed with a Micro Fiber TensileMachine, outfitted with a 35 N load cell and a special stainless steelgrips. The grips clamped the specimens by jaws machined from Delrin(acetal). The control of machine was performed by developed software onMatlab6 and data acquisition performed with National Instrumentshardware on a PentiumII PC. The load cell was zeroed after theattachment of each sample. Samples were moistened by regularly applyingdrops of PBS. The gauge length was taken to be the length of theconstruct between the grips, which was measured from the calibratedimages of a fast MotionScope CCD camera. Tests were conducted at aconstant displacement rate of 0.08 mm/sec.

In-vVivo Studies—

For subcutaneous transplantation (ectopic transplantation), cd1 nudemice were anesthetized and approximately 5 mm long incision was made in2 or 3 locations on the back of the animal. CTPs from fat (from passage12-14) and fetal (from passages 5-6 and 10-11) tissues were implantedinto the subcutaneous pouch. The method for critical Achilles tendoninjury model preparation is described in the description of FIGS. 13a-e. All animal experimental protocols were approved by the Animal Useand Care Committee of the Technion Faculty of Medicine.

In Vivo Transplantation of CTPs Underneath Kidney Capsules—

Subconfluent CTP cultures (from fetal derived CTPs, passages 5-6 and10-11) were scraped off the culture plates, washed with PBS andimplanted underneath the kidney capsule of 5-week-old CB-17 SCID-beigemice (n=6). Six and twelve weeks after transplantation, kidneys wereretrieved, fixed in 10% buffered formalin, embedded in paraffin, andsectioned for histological examination.

Example 1 Isolation of Connective Tissue Progenitor Cells From HumanAdult Stem Cells

A cell source for tissue engineering should be highly proliferativewhile phenotypically stable in vitro, providing a sufficient amount ofcells. As estimated elsewhere (Muschler, G. F. et al, 2002),approximately 7×10⁷ osteoblasts are needed to form one cubic centimeterof a new bone. While reducing the present invention to practice, thepresent inventors have uncovered, through laborious experimentations,culturing conditions suitable for the isolation, propagation anddifferentiation of connective tissue progenitor cells derived from adultstem cells, as follows.

Experimental Results

Isolation of Connective Tissue Progenitor Cells (CTPs) From Adult StemCells—

Adult stem cells from fat, foreskin or fetal tissue were dissociatedwith a combination of enzymatic digestion and aggressive mechanicalagitation. The derived cells were cultured in the presence of a CTPmedium containing ascorbic acid and dexamethasone, factors known topromote osteoblastic differentiation (Maniatopoulos, C., et al, 1988;Coelho, M. J. et al, 2000). The cells were cultured without splitting(passaging) in the presence of the CTP medium until reachingsub-confluency (between about 5-10 days) and were then subjected toserial passaging using type IV collagenase (0.1%, 30-60 minutes at 37°C.) followed by trypsinization (0.25%, 2-5 minutes at 37° C.). Thegrowth kinetic pattern of the cells, which were continuously expandingwithout obvious senescence up to 25 passages, suggested the derivationof a putative cell line. At passage 25 the growth rate of the cells wasslowed down (data not shown) and the cells tended to form mineralizedcultures at a higher frequency (data not shown). Cultures recovered wellafter freeze/thaw cycles, and showed the same proliferation anddifferentiation potential as prior to the freeze/thaw cycles. Inaddition, adult stem cell derived CTPs from early passages exhibit amorphology resembling that of mesenchymal stem cells (MSCs, FIGS. 3 a-b)and CTPs from later passages exhibit a morphology resembling that offibroblasts (FIG. 3 c).

CTPs Exhibit Clonogenic Potential and Normal Karyotype—

The clonality potential of the fetal derived CTP cells was assessed byseeding single cells each in different culture wells. Colonies wereformed in high efficiency (20-22 out of 24 experiments (data not shown)and were identical in their growth kinetics, morphology and phenotype,as assessed by RT-PCR. Additionally, G banding analysis of fat stemcells derived CTP nuclei obtained from passages 5-6 and from fetalderived CTPs from passages 7-8, 14 and 20 confirmed that the CTPs arekaryotypically normal (data not shown).

CTPs are Suitable for Cell-Based Tissue Engineering—

Assuming symmetric cell division, at least 3.5×10⁹ connective tissueprogenitor cells are obtained from a single cell of the adult stem cellsfollowing about 20 passage. Thus, the unique derivation method of thepresent invention generates a sufficient amount of cells for cell-basedtissue engineering application.

Altogether, the present inventors have demonstrated that the CTP cellsisolated from adult stem cells by culturing and passaging in the CTPmedium exhibit high proliferative capacity (for at least 20 passages),in vitro stability (with normal karyotype) and clonogenic potential. Inaddition, the present inventors have uncovered that the CTPs of thepresent invention are suitable for cell-based tissue engineering.

Example 2 CTPs Express Markers of Mesenchymal and Osteoblast-Like Cells

To test the potential of the adult stem cells derived CTPs of thepresent invention to differentiate into the osteogenic lineage, theexpression profile of the CTP cells of the present invention wasassessed by RT-PCR, cDNA microarray and FACS analyses, as follows.

Experimental Results

CTPs Express Osteogenic Markers—

Gene expression of CTP cells grown in the presence of the CTP mediumfrom passages 1 and 10, was assessed by RT-PCR analysis. As is shown inFIGS. 2 a-u, CTPs stably express core binding factor alpha 1 (CBFA1),and SOX9, both are early transcription factors known to play a majorrole in osteoblast and chondrocyte differentiation. Type I collagen, themost abundant extracellular protein which is synthesized by osteoblasts,osteonectin and osteopontin, two major non-collagenous bone matrixproteins, parathyroid hormone receptor 1 (PTHR1), which regulatesmineral homeostasis and bone formation, and bone-specific alkalinephosphatase, which binds phosphor to calcium and forms bonehydroxyapetite, were all detected in the CTPs, indicating osteogenicpotential.

Immunofluorescence analysis of the adult stem cells derived CTPsdemonstrated that the fetal-derived CTPs of the present inventionexpress alkaline phosphatase (ALP, FIG. 7 a) and osteocalcin (FIG. 7 b),markers of the osteogenic lineage.

Expression Profiles of CTP-Derived Osteogenic-Like Cells—

The gene expression profile of the cells was detected using cDNAmicroarray analysis using a set of approximately 100osteogenesis-related genes. The common transcripts were compared betweenhESC-derived CTPs (passage 1 and 9) to human fetal CTPs (passage 1). Twoindependent experiments were performed. The complete list of positivetranscripts is shown in Table 2, hereinbelow. Forty transcripts werefound to be shared among hESC-derived CTPs and human fetal CTPs,including growth factors and associate molecules, cell adhesionmolecules, and matrix associated proteins. A significant overlap of 36transcripts shared by the three populations (i.e., undifferentiatedhESCs, hESC-derived CTPs and human fetal CTPs) was observed. This set ofshared transcripts represent genes that though known to play asignificant role in osteogenesis, are already switched-on at the hESCstage.

TABLE 2 Expression profile of fetal stem cells-derived CTPs incomparison to undifferentiated hESCs and hESCs-derived CTPs (p9) SymbolGene Name Undiff- hESCs CTP, p9 Fetal Description ALPL AP-TNAP/HOPS p pn Alkaline phosphatase, liver/bone/kidney ANXA5 ANX5/ENX2 p p p AnnexinA5 ARSE CDPX/CDPX1 n n n Arylsulfatase E (chondrodysplasia punctata 1)BGLAP BGP n n n Bone gamma- carboxyglutamate (gla) protein (osteocalcin)BGN DSPG1/PG-S1 p p p Biglycan BMP1 PCOLC/TLD n p n Bone morphogeneticprotein 1 BMP2 BMP2A p p n Bone morphogenetic protein 2 BMP3 BMP3 p p nBone morphogenetic protein 3 (osteogenic) BMP4 BMP2B/BMP2B1 n n n Bonemorphogenetic protein 4 BMP5 MGC34244 p p p Bone morphogenetic protein 5BMP6 VGR/VGR1 n n n Bone morphogenetic protein 6 BMP7 OP-1 p p p Bonemorphogenetic protein 7 (osteogenic protein 1) BMP8B BMP8/OP2 p p p Bonemorphogenetic protein 8b (osteogenic protein 2) BMPR1A ACVRLK3/ALK3 p pp Bone morphogenetic protein receptor, type IA CASR FHH/FIH n n nCalcium-sensing receptor (hypocalciuric hypercalcemia 1, severe neonatalhyperparathyroidism) CD36 FAT/GP3B n n n CD36 antigen (collagen type Ireceptor, thrombospondin receptor) SCARB1 CD36L1/CLA-1 p p p Scavengerreceptor class B, member 1 CSIG L12/PBK1 n n n Ribosomal L1 domaincontaining 1 COL10A1 COL10A1 p p n Collagen, type X, alpha 1(Schmidmetaphyseal chondrodysplasia) COL11A1 CO11A1/COLL6 n p p Collagen, typeXI, alpha 1 COL12A1 COL12A1L/TYPE n p p Collagen, type XII, XII alpha 1COL14A1 UND p p n Collagen, type XIV, alpha 1 (undulin) COL15A1 COL15A1n n n Collagen, type XV, alpha 1 COL16A1 447AA/FP1572 p p p Collagen,type XVI, alpha 1 COL17A1 BP180/BPAG2 n p n Collagen, type XVII, alpha 1COL18A1 KNO p p p Collagen, type XVIII, alpha 1 COL19A1 COL9A1L/D6S228En n n Collagen, type XIX, alpha 1 COL1A1 AA 694-711/OI4 n n n Collagen,type I, alpha 1 COL2A1 COL11A3/SEDC p p p Collagen, type II, alpha 1(primary osteoarthritis, spondyloepiphyseal dysplasia, congenital)COL3A1 EDS4A p p p Collagen, type III, alpha 1 (Ehlers-Danlos syndrometype IV, autosomal dominant) COL4A3 TUMSTATIN n n n Collagen, type IV,alpha 3 (Goodpasture antigen) COL4A4 CA44 p p p Collagen, type IV, alpha4 COL4A5 ASLN/ATS n n n Collagen, type IV, alpha 5 (Alport syndrome)COL5A1 COL5A1 p p p Collagen, type V, alpha 1 COL7A1 EBD1/EBDCT n p nCollagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominantand recessive) COL9A2 EDM2/MED p p p Collagen, type IX, alpha 2 CSF2GMCSF p n n Colony stimulating factor 2 (granulocyte- macrophage) CSF3G-CSF/GCSF n n n Colony stimulating factor 3 (granulocyte) CTSKCTS02/CTSO n n p Cathepsin K (pycnodysostosis) DCN DSPG2/PG40 p p pDecorin EGF URG n n n Epidermal growth factor (beta- urogastrone) EGFRERBB/ERBB1 p p n Epidermal growth factor receptor (erythroblasticleukemia viral (v-erb- b) oncogene homolog, avian) FGF1 AFGF/ECGF n p nFibroblast growth factor 1 (acidic) FGF2 BFGF/FGFB p p n Fibroblastgrowth factor 2 (basic) FGF3 HBGF-3/INT2 n n n Fibroblast growth factor3 (murine mammary tumor virus integration site (v-int- 2) oncogenehomolog) FGFR1 BFGFR/C-FGR p p p Fibroblast growth factor receptor 1(fms- related tyrosine kinase 2, Pfeiffer syndrome) FGFR2 BEK/BFR-1 p nn Fibroblast growth factor receptor 2 (bacteria-expressed kinase,keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzonsyndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) FGFR3 ACH/CEK2 p nn Fibroblast growth factor receptor 3 (achondroplasia, thanatophoricdwarfism) FLT1 FLT/VEGFR1 p n n Fms-related tyrosine kinase 1 (vascularendothelial growth factor/vascular permeability factor receptor) FN1CIG/FINC p p p Fibronectin 1 GDF10 BMP-3B/BMP3B p p n Growthdifferentiation factor 10 1CAM1 BB2/CD54 n p n Intercellular adhesionmolecule 1 (CD54), human rhinovirus receptor IGF1 IGFI n p pInsulin-like growth factor 1 (somatomedin C) IGF1R JTK13 p p nInsulin-like growth factor 1 receptor IGF2 IGF-II n n n Insulin-likegrowth factor 2 (somatomedin A) ITGA1 CD49a p p p Integrin, alpha 1ITGA2 BR/CD49B n n n Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2receptor) ITGA3 CD49C/GAP-B3 p p n Integrin, alpha 3 (antigen CD49C,alpha 3 subunit of VLA-3 receptor) ITGAM CD11B/CR3A n n n Integrin,alpha M (complement component receptor 3, alpha; also known as CD11b(p170), macrophage antigen alpha polypeptide) ITGAV CD51/MSK8 p p pIntegrin, alpha V (vitronectin receptor, alpha polypeptide, antigenCD51) ITGB1 CD29/FNRB p p p Integrin, beta 1 (fibronectin receptor, betapolypeptide, antigen CD29 includes MDF2, MSK12) SMAD1 BSP1/JV4-1 p p pSMAD, mothers against DPP homolog 1 (Drosophila) SMAD2 MADH2/HSMAD2 p pp SMAD, mothers against DPP homolog 2 (Drosophila) SMAD3HSPC193/HST17436 p p n SMAD, mothers against DPP homolog 3 (Drosophila)SMAD4 DPC4/JIP p p p SMAD, mothers against DPP homolog 4 (Drosophila)SMAD5 DWFC/JV5-1 p p p SMAD, mothers against DPP homolog 5 (Drosophila)SMAD6 HST17432/MAD p p n SMAD, mothers against DPP homolog 6(Drosophila) SMAD7 MAD/MADH7 p p n SMAD, mothers against DPP homolog 7(Drosophila) SMAD9 MAD/MADH6 p p p SMAD, mothers against DPP homolog 9(Drosophila) MMP10 SL-2/STMY2 n n n Matrix metalloproteinase 10(stromelysin 2) MMP13 CLG3 p p p Matrix metalloproteinase 13(collagenase 3) MMP2 CLG4/CLG4A p p p Matrix metalloproteinase 2(gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) MMP8CLG1/HNC n n p Matrix metalloproteinase 8 (neutrophil collagenase) MMP9GELB/CLG4B n n p Matrix metalloproteinase 9 (gelatinase B, 92 kDagelatinase, 92 kDa type IV collagenase) MSX1 HOX7/HYD1 p n n Msh homeobox homolog 1 (Drosophila) MSX2 CRS2/FPP n n n Msh homeo box homolog 2(Drosophila) NFKB1 EBP-1/KBF1 n p p Nuclear factor of kappa lightpolypeptide gene enhancer in B-cells 1 (p105) PDGFA PDGF-A/PDGF1 p p nPlatelet-derived growth factor alpha polypeptide RUNX2 CCD/AML3 p p pRunt-related transcription factor 2 SERPINH1 ASTP3/CBP1 p p p Serine (orcysteine) proteinase inhibitor, clade H (heat shock protein 47), member1, (collagen binding protein 1) SERPINH1 ASTP3/CBP1 p p p Serine (orcysteine) proteinase inhibitor, clade H (heat shock protein 47), member1, (collagen binding protein 1) SOX9 CMD1/CMPD1 n n n SRY (sexdetermining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal) SPARC ON p p p Secreted protein, acidic, cysteine-rich(osteonectin) SPP1 BNSP/BSPI p n n Secreted phosphoprotein 1(osteopontin, bone sialoprotein I, early T- lymphocyte activation 1)TGFB1 BETA 1/CED n p n Transforming growth factor, beta 1(Camurati-Engelmann disease) TGFB2 TGF b2 n p n Transforming growthfactor, beta 2 TGFB3 TGF b3 p p p Transforming growth factor, beta 3TGFBR1 ACVRLK4/ALK-5 p p p Transforming growth factor, beta receptor I(activin A receptor type II-like kinase, 53 kDa) TGFBR2 HNPCC6/MFS2 n pn Transforming growth factor, beta receptor II (70/80 kDa) TNFDIF/TNF-ALPHA p p p Tumor necrosis factor (TNF superfamily, member 2)TWIST1 ACS3/BPES2 p p n Twist homolog 1 (acrocephalosyndactyly 3;Saethre-Chotzen syndrome) (Drosophila) VCAM1 INCAM-100 n n n Vascularcell adhesion molecule 1 VDR NR1I1 n n n Vitamin D (1,25-dihydroxyvitamin D3) receptor VEGF VEGFA p p p Vascular endothelialgrowth factor VEGFB VEGFL/VRF n n n Vascular endothelial growth factor BVEGFC FLT4-L/VRP p p p Vascular endothelial growth factor C PUC18 pUC18n n n PUC18 Plasmid DNA PUC18 pUC18 n n n PUC18 Plasmid DNA PUC18 pUC18n n n PUC18 Plasmid DNA n n n n n n n n n GAPDH G3PD/GAPD p p pGlyceraldehyde-3- phosphate dehydrogenase GAPDH G3PD/GAPD p p pGlyceraldehyde-3- phosphate dehydrogenase PPIA CYPA/CYPH p p pPeptidylprolyl isomerase A (cyclophilin A) PPIA CYPA/CYPH p p pPeptidylprolyl isomerase A (cyclophilin A) PPIA CYPA/CYPH p p pPeptidylprolyl isomerase A (cyclophilin A) PPIA CYPA/CYPH p p pPeptidylprolyl isomerase A (cyclophilin A) RPL13A RPL13A p p p Ribosomalprotein L13a RPL13A RPL13A p p p Ribosomal protein L13a ACTB b-Actin p pp Actin, beta ACTB b-Actin p p p Actin, beta Table 2: The cDNA arraymembrane (GEArray Q Series Human Osteogenesis Gene Array HS-026;SuperArray Bioscience Corp., Frederic, MD USA) was hybridized withbiotin-labeled cDNA prepared from human fetal CTPs (fetal, passage 1),undifferentiated hESCs (H9.2; undiff-hESCs), and hESC-derived CTPs atpassage 1 (p1) or passage 9 (p9). The presence (positive, “p”) orabsence (negative, “n”) of each of the genes in the array is indicated.

Adult Stem Cells—Derived CTPs are CD105/CD166/CD44/CD29/HLA-ABC-Positiveand CD45/HLA-DR-Negative—

FACS analyses performed on adult stem cells derived CTPs from passage 16using antibodies specific to the CD105, CD166, CD44, CD29, CD45, HLA-ABCand HLA-DR surface markers demonstrated relatively high level ofpopulation purity with surface markers characteristic of MSCs (FIGS. 1a-g). Thus, the CTPs of the present invention were positive for CD105,CD166, CD44, CD29 and HLA-ABC, while negative for CD45, a hematopoieticmarker and HLA-DR.

Altogether, the FACS results demonstrate that adult stem cells-derivedCTPs express markers of mesenchymal stem cells (MSCs). The cDNAmicroarray and the RT-PCR analyses demonstrate the presence of specificmarkers of connective tissue derivatives including osteogenic lineage,chondrogenic lineage, tendons and ligaments. In addition, immunostaininganalyses demonstrated that the CTP cells isolated according to theteachings of the present invention express markers of the osteogeniclineage and thus can differentiate to form osteoblasts and bone tissue.

Example 3 CTPs are Capable of Producing Bone Matrix

To test to capacity of the CTPs of the present invention (from fetaltissue, foreskin tissue and fat tissue) to differentiate into theosteogenic lineage and form mineralized matrix, inorganic phosphate wasadded to the CTP medium and the CTP cell cultures were assessed for thepresence of mineralized matrix, as follows.

CTP Cells Produce Mineralized Matrix—

To test the capacity of the CTP cells of the present invention to formmineralized matrix, inorganic phosphate (beta-glycerophosphate) wasadded to the CTP medium. Briefly, CTPs from any passage of passages 1-25were grown in a CTP medium supplemented with 10 mM beta-glycerophosphateand the potential of matrix formation was assessed. As is shown in FIG.8 a, enhanced mineralization which was visible macroscopically (e.g., atpassage 11), or was induced at different passages if cells were grownuntil over-confluent. Mineralization was confirmed by Alizarin Redstaining (FIG. 8 b), and scanning electron microscopy (FIG. 8 d) coupledwith EDS spectra analysis of the bone mineralization. The EDS analysisdetected calcium and phosphate as the most prominent signals, inaddition to carbon and oxygen which could correspond to the presence ofproteins. The ratio between the calcium and phosphate corresponds to theexpected ratio of the hydroxyapatite mineral (data not shown). Moreover,immunostaining analysis demonstrated the presence of self-producedmatrix which is positive for type I collagen (FIG. 8 c). Thus, theseresults demonstrate that the CTPs of the present invention are capableof forming bone nodules containing calcium-phosphate deposits, the majorcomponent of bone minerals.

Altogether, these results demonstrate that adult stem cells-derived CTPsare capable of differentiating into cells of the osteogenic lineagewhile producing mineralized matrix.

Example 4 Human Adult Stem Cells-Derived CTP Cells are Capable ofDifferentiating Into the Chondrogenic Lineage

To induce to chondrogenic differentiation, the two differentiationmethods described under “General Materials and Experimental Methods”were employed.

Experimental Results

Induction of CTPs to Chondrogenic Differentiation Using the “IntactLayer” Method—

To induce chondrogenic differentiation, subconfluent cultures (passages5-13) were trypsinized and placed as an intact layer in suspension, inthe presence of the CTP medium. After 10 days the tissue was harvestedfor analysis. H&E staining showed round chondrocyte-like cells embeddedin lacunae, at the periphery of the sample (FIG. 9 a). Picro-sirius redstaining demonstrated extensive collagenous matrix surrounding the cells(FIG. 9 b).

Induction of CTPs to Chondrogenic Differentiation Using the PelletCulture Method—

The induction of chondrogenic differentiation was further assessed usingthe pellet culture system of chondrogenic differentiation as publishedelsewhere (24). Cells (passages 1-9, of fetal derived CTPs, and passages5, 10, 15-20, of fat-derived CTPs) were grown as pellet cultures in thepresence of low serum TGF-β3 supplemented medium as described in method2 of chondrogenic differentiation in the General Materials andExperimental Methods, hereinabove. A section of one-month-old pelletculture was stained with Toluidine blue, showing the matrixproteoglycans (FIG. 10). Earlier pellet cultures were not positivelystained (data not shown).

Altogether, these results demonstrate that adult stem cells-derived CTPsare capable of differentiating into the chondrogenic lineage whileexpressing cartilage markers.

Example 5 Enhanced Matrix Production by CTPS Seeded on NanofiberScaffolds

To provide cells with a suitable 3D environment that would support theirgrowth and organization into a complex tissue, the electrospun, PCL/PLAnanofiber scaffolds were utilized, as follows.

Experimental Results

Seeding of CTPs on Electrospun PCL/PLA Scaffolds Resulted in ConnectiveTissue Formation—

5×10⁵ CTPs (passage 10-12) resuspended in 10 μl of CTP medium wereseeded on scaffolds and grown in the presence of the CTP medium for onemonth before harvesting for analysis. Histological analysis showed anhigh matrix to cell ratio (ECM-rich) connective tissue formation, withmesenchymal-like cells aligned in parallel, surrounded with extensivefibrous collagen matrix, with a thin remnant of the scaffold at thebasal side of the construct, suggesting it has degraded over time (datanot shown). Scanning electron microscopy illustrated the beginning ofmatrix production and a progressive tissue-like patches formation untila firm 3D sheet-like tissue is generated (data not shown). The presenceof mineral deposits was confirmed by EDS analysis (data not shown).Cells grown in the same conditions but on plastic tissue culture platesdid not show any form of 3D tissue-like formation (data not shown).

Altogether, these results demonstrate the ability of the adult stemcells derived CTPs of the present invention to form mineralized matrixand tissue-like patches when grown on nano-scaffolds.

Example 6 CTPS Are Capable of Forming Tendon Grafts in Vitro

To test the ability of the adult stem cells derived CTPs of the presentinvention to form a more compact and organized tissue, the long-termhigh-density culture technique was optimized, as follows.

CTP Cells are Capable of Forming a Tendon-Like Tissue—

The long-term high-density culture technique induces the self-assemblyof 3D, cylinder-shaped constructs, which morphologically resemble earlydeveloped tendons. This process begins as a single cell attaching to theculture plate sidewall, forming fibrous matrix with a tendon-like shape.Adult stem cells derived CTPs from either a fat tissue (FIGS. 5 a-f) ora fetal tissue (FIGS. 5 g-m) which were cultured in the presence of CTPmedium were capable of forming a tendon-like construct following 8 weeksin culture and a clear tendon tissue which grew up to 5 centimetersfollowing 4 months in culture or 6-8 cm following 4-5 months (data notshown). Further immunostaining analyses demonstrated the progressiveassembly of long, cylinder-shaped constructs which express type Icollagen (data not shown). At a later developmental stage (following 2-3weeks in culture), wider structures were observed (FIGS. 5 c-e), untilthe formation of well defined, tendon-like constructs (FIG. 5 f).Histological examination shows organized, parallel-aligned cells, withhigh matrix-to-cell ratio (data not shown). Additional SEM analyses ofthe tendon graft demonstrated well-defined fibrous structure andparallel orientation of elongated, fibroblast-like looking, cells (FIG.5 i, j and data not shown). Thus, these cells formed a well organized,highly cellular collagen-associated mineralized tissue, as evident byhistology and electron microscopy (FIG. 5 k, l, m and data not shown).Altogether, these experiments demonstrate the engineering of functionaltendons from the adult stem cells derived CTPs of the present invention.

Evaluation of the Mechanical Properties of the Engineered ConstructsDerived from the CTP Cells—

Three-month old constructs (made from fetal derived CTPs) were removedfrom the culture plate and were put into a custom built testing machine.The strain-stress response of the constructs resembles a non-linearbehavior.

Altogether, these results demonstrate the formation of a tendon-likestructure with excellent mechanical properties resembling that of maturerather than immature tendons.

Example 7 Adult Stem Cells Derived CTP Cells Form Connective Tissue InVivo

To assess the commitment of the adult stem cells-derived CTP cells tothe connective tissue lineage on one hand, and to examine theirtumorigenic properties on the other hand, CTPs were transplanted into 6SCID-beige mice, as follows.

CTP Cells are Non-Tumorigenic in Vivo—

Fetal derived CTPs from passages 5-6 or 10-11 were implanted underneaththe kidney capsule of 5-week-old SCID-beige mice. Using this animalmodel, the present inventors have previously shown that undifferentiatedhESCs are capable of forming teratomas (data not shown). Six and twelveweeks post transplantation, kidneys were harvested for analysis. Bothmacroscopic examination and histological analysis did not detect anyform of teratoma formation (data not shown). Instead, a localizedsub-capsular formation of loose connective tissue was observed, with noevidence of other tissue type (data not shown). Using this model, it ispossible to explore the in-vivo developmental potential of adult stemcells-derived CTPs.

Altogether, these results demonstrate that adult stem cells-derived CTPsare non-tumorigenic and are committed to the connective cell lineage invivo.

Example 8 CTP Cells Form Bone and Cartilage Tissues In Vivo

To assess the potential of the adult stem cells derived CTPs of thepresent invention to form cartilage and bone tissue in vivo, CTPs weresubcutaneously transplanted into cd1 nude mice and the formation ofectopic new bone and cartilage was assessed, as follows.

CTP Derived Ectopic Transplants are Biocompatible, Well-VascularizedMasses with Radio-Opaque Bone Tissue—

For subcutaneous transplantation of adult stem cells derived CTPs, themice were anesthetized and approximately 5 mm long incisions were madein 2 or 3 locations on the back of the animal. CTP cells from a singleT75 flask (from either fetal or fat-derived CTPs) were injected in eachincision. Ectopic transplants visible following 3 weeks oftransplantation included bone tissue, as confirmed by the radio-opaquetransplants seen by X-ray analysis (FIG. 4 a and data not shown).Furthermore, X-ray analysis performed following 5 months oftransplantation confirmed the formation of bone tissue (FIGS. 4 b andc). It should be noted that the presence of bone tissue following 5months of transplantation demonstrates that the CTPs underwent terminaldifferentiation to bone tissue and that the transplantation was notassociated with a tumorigenic process. Close examination of the ectopictransplants following the removal of the back skin revealed the presenceof non-cancerous, well-vascularized masses (FIGS. 11 a-c), demonstratingthat the ectopic transplant is biocompatible, well integrated within therecipient mouse and not rejected by its immune system.

CTP Derived Ectopic Transplants Include Bone and Cartilage Tissues—

Histological analyses of the ectopic transplants revealed the formationof new bone from CTPs derived from fetal tissue (data not shown) andhypertrophic cartilage (data not shown) tissues with early stage ofmineralized bone matrix formation and a later stage showing osteon-likestructures and new bone formation (data not shown).

CTP Derived Ectopic Transplants Include Mineralized Bone Tissue—

As is further shown in FIG. 12 a, analysis of frozen sections ofnon-demineralized tissue demonstrated the formation of mineral depositswithin the ectopic transplants of the adult stem cells derived CTPs(from fat tissue). In addition, H&E staining of histological sectionsderived from the ectopic transplant confirmed the formation of bonetissue (FIG. 12 b).

CTP Derived Ectopic Transplants are of a Human Origin—

To verify that the ectopic transplants are derived from the adult stemcells derived CTPs from a fetal tissue histological sections of theectopic transplants were subjected to immunostaining analysis using theanti-human mitochondria antibody (BioGenex, San Ramon, Calif., USA, Cat.No MU213-UC). The newly-formed ectopic tissue is positively stained withthe anti-human mitochondria antibody (data not shown), demonstrating thehuman origin of the ectopic tissue.

Altogether, these results demonstrate the ability of the adult stemcells derived CTPs of the present invention to form bone and cartilagetissue in vivo.

Example 9 The In Vivo Repair of Critical Achilles-Tendon Injury Using aTendon Graft Formed from Adult Stem Cells Derived CTPS

To test the functionality of the in vitro engineered tendon graft fromthe adult stem cells derived CTPs of the present invention (as describedin Example 6, hereinabove), the present inventors have induced acritical Achilles-tendon injury in mice and implanted the tendon graftof the present invention, as follow.

FIGS. 13 a-e schematically depict the strategy of repairing a criticalAchilles-tendon injury using the in vitro generated tendon graft of thepresent invention. High-density CTP cultures were grown with no furthersplitting for 4-5 weeks to form a sheet-like tissues in culture plates(FIG. 13 a). Next tissues were gently removed from plates using a cellscraper and rolled to form rounded cylinders (FIG. 13 b). Non-absorbablesutures were inserted at the ends of a construct through all layers(FIG. 13 c). Constructs were immediately used for transplantation orkept inside custom-made templates made from flexible silicon tubesembedded in agar plates. Full thickness, 3-4 mm long segment of theAchilles tendon in nude mice was cut to form a critical gap (FIG. 13 d).Next constructs were sutured to the proximal and distal edges of theinjured Achilles tendon (FIG. 13 e).

Experimental Results

In Vivo Repair of a Critical Achilles-Tendon Injury Using the in VitroFormed Tendon Graft of the Present Invention—

Following the induction of a critical Achilles-tendon injury the treatedmice are unable to extent their ankle, resulting in a maximal extensionof less than 90 degrees (FIG. 14 a). To repair the injury, adult stemcells derived CTPs from fetal and adult tissues are subjected to ahigh-density culture to form tendon grafts which are then implanted inthe injured mice (FIG. 14 b). One month after transplantation theimplanted mice are capable of extending their leg with a maximalextension greater than 90 degrees (data not shown).

The Transplanted Tendon Grafts are Remodeled and Exhibit ExcellentBiomechanical Properties—

Following 6-8 weeks of transplantation, the transplanted tendon graftsexhibit good integration at the sites of suturing (data not shown), withcirculating blood vessels invading the graft (data not shown). Furtherhistological evaluation of the transplanted grafts (generated from fetalderived CTPs) demonstrated that the grafts remodeled and integrated well(data not shown) with smoother edges. Further biomechanical testingusing the tensile test machine demonstrated that the stress/strainbehavior of the tendon graft (generated from fetal derived CTPs) aftertransplantation resembles that of a native rat tail tendon (data notshown).

The Transplanted Tendon Grafts are of a Human Origin—

To confirm the human origin of the transplanted tendon grafts (formedfrom fetal derived CTPs), a cross section of the transplanted tendon wassubjected to immunostaining analysis using the anti-human mitochondriaantibody. The transplanted tendon is positively stained with theanti-human mitochondria antibody, demonstrating its human origin (datanot shown).

Example 10 Formation of ECM and Accelularized Matrix Using theConnective Tissue Progenitor Cells

Experimental Results

Formation of ECM in Vitro—

For the formation of extracellular matrix (ECM), the CTPs (from fattissue) were plated at high densities (5-10×10⁵ cells/cm²) in tissueculture plates in the presence of a culture medium comprising ascorbicacid and dexamethasone (CTP medium). After about 4 weeks in culture(other periods of time are also possible) sheet-like tissue was formed.The sheet-like tissue was subjected to freeze-drying and/or cell removal(FIGS. 15 a-j). Freeze-drying was done using standard lyophilizationdevice. The resultant tissue was completely devoid of viable cells asdetecting using e.g., standard viability assays, thus reducing the riskof cell-based tumor formation and immune rejection.

Dry ECM Tissues are Biocompatible—

Dry tissues were stored at room temperature in dry conditions. The drytissue formed was found to be biocompatible as evidenced by seeding thedry tissues with different cell types (e.g., Human embryonic kidney 293cells) and observing the formation of secondary new tissue, as a modelfor allogenic cell seeding (FIGS. 16 a-h, 17 and 18 a-d).

The dried tissues were characterized by histology, immunostaining,electron microscopy and the results demonstrated that the ECM was keptintact, in a native form, supporting biological cellular activities(FIGS. 16 a-f, 16 g-h).

Acellularization of ECM—

Cell removal was done chemically with detergents such as SDS (0.1%,incubation time 20-60 minutes), combined with protease inhibitors,essentially as described in Cartmell J S. and Dunn M G., 2000, J. BiomedMater. Res. 49(1): 134-40. Histology and electron microscopy assays showthat the resultant tissue is completely acellular, while the ECM ispreserved (FIGS. 16 a, 16 c, 16 e, 18 a-f).

Acellularized ECM Tissue is Biocompatible—

The biocompatibility of the acellular tissues was demonstrated byseeding different cell types onto the tissue, with the formation ofsecondary new tissue, as a model for allogenic cell seeding (FIGS. 16 b,16 d, 16 f, and 18 a-i).

In addition, as is shown in FIGS. 19 a-f, when transplanted in vivo theacellularized tissue (generated from fetal derived CTPs) isbiocompatible as evidenced by histological staining of the tissue oneweek after transplantation with no signs of graft rejection.

Altogether, the freeze-dried and/or de-cellularized CTP-derived tissuesare easy to handle and shape, suture retentive, can rapidly re-hydrate(indicates high hydrophilicity) and can be combined with standardhydrogels to make an injectable form.

It will be appreciated that the intact ECM can be conditioned byspecific cell types, such as skin, skeletal muscle, cardiac muscle, fat,cartilage, bone, etc., to create tissue specific, specialized intact ECMtissues. This can be done by co-culturing CTPs with the second cell typebefore the formation of the primary tissue, or by seeding the secondcell type onto the already processed CTP-derived tissue, allowing thecells to interact with the intact-ECM tissue, before secondary round offreeze-drying and/or cell removal will occur. Alternatively, the secondcell type taken from the patient can be left to grow on the tissue priorto transplantation back into the patient.

Purified ECM Components—

Total protein component or selected proteins is purified from CTPs orCTP-derived tissues according to standard protein purification methods.The resultant protein is assessed by gel electrophoresis, westernblotting and proteomics analysis. Such proteins are processed in orderto achieve clinical-grade injectable formulas and other forms suitablefor biomaterial applications.

Example 11 Adult Stem Cells Derived CTPS are Capable of Forming FatTissue

To further test the potential of CTPs which were generated fromfat-tissue stem cells to form fat tissue, CTPs derived from any passage1-20 were cultured in a culture medium containing αMEM supplemented with15% FBS (selected lots), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10μg/ml insulin, 10⁻⁶ M dexamethasone, and following 7-21 days in cultureadipocytes were observed (FIG. 6 a). To further test the potential ofCTPs derived from another adult tissue to differentiate into the fatcell lineage, fetal derived CTPs were cultured in the same culturemedium described hereinabove and following 7-14 days in cultureadipocytes were observed (FIG. 6 b).

Altogether, these results conclusively show that adult stemcells—derived CTPs can differentiate into fat tissue and formadipocytes.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications and GenBank Accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application or GenBank Accession numberwas specifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

REFERENCES Additional References are Cited in Text

-   1. Vacanti, J. P. & Langer, R. (1999) Lancet 354, SI32-SI34.-   2. Sharma, B. & Elisseeff, J. H. (2004) Ann Biomed Eng. 32, 148-159.-   3. Muschler, G. F., Nakamoto, C. & Griffith, L. G. (2004) J Bone    Joint Surg Am. 86, 1541-1558.-   4. Lavik, E. & Langer, R. (2004) Appl Microbiol Biotechnol 65, 1-8.-   5. Betz, R. R. (2002) Orthopedics. 25, s561-s570.-   6. Meyer, U., Joos, U. & Wiesmann, H. P. (2004) Int J Oral    Maxillofac Surg 33, 325-332.-   7. Muschler, G. F. & Midura, R. J. (2002) Clin Orthop Relat Res    66-80.-   8. Sottile, V., Halleux, C., Bassilana, F., Keller, H. &    Seuwen, K. (2002) Bone 30, 699-704.-   9. Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K.,    Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig,    S, & Marshak, D. R. (1999) Science 284, 143-147.-   10. Mastrogiacomo, M., Derubeis, A. R. & Cancedda R. (2005) J Cell    Physiol 204, 594-603.-   11. Zuk, P. A., Zhu, M., Mizuno, H., Huang, J., Futrell. J. W,    Katz, A. J., Benhaim, P., Lorenz, H. P. & Hedrick, M. H. (2001)    Tissue Eng 7, 211-228.-   12. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M.    A., Swiergiel, J. J., Marshall, V. S. & Jones, J. M. (1998) Science    282, 1145-1147.-   13. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A.,    Yanuka, O., Amit, M., Soreq, H. & Benvenisty, N. (2000) Mol Med 6,    88-95.-   14. Schuldiner, M., Yanuka. O., Itskovitz-Eldor. J., Melton, D. A. &    Benvenisty, N. (2000) Proc Natl Acad Sci USA 97, 11307-11312.-   15. Hwang, W. S., Roh, S. I., Lee, B. C., Kang, S. K., Kwon, D. K.,    Kim, S., Kim, S. J., Park, S. W., Kwon, H. S., Lee, C. K. et    al. (2005) Science 308, 1777-1783.-   16. Levenberg, S., Huang, N. F., Lavik, E., Rogers, A. B.,    Itskovitz-Eldor, J. & Langer, R. (2003) Proc Natl Acad Sci USA. 100,    12741-12746.-   17. Gerecht-Nir, S., Cohen, S., Ziskind, A. & Itskovitz-Eldor,    J (2004) Biotechnol Bioeng. 88, 313-320.-   18. Cao, T., Heng, B. C., Ye, C. P., Liu, H., Toh, W. S., Robson,    P., Li, P., Hong, Y. H. & Stanton, L. W. (2005) Tissue Cell 37,    325-334.-   19. Bielby, R. C., Boccaccini, A. R., Polak, J. M. &    Buttery L. D. (2004) Tissue Eng 10, 1518-1525.-   20. Sottile, V., Thomson, A. & McWhir, J. (2003) Cloning Stem Cells    5, 149-155.-   21. Barberi, T., Willis, L. M., Socci, N. D. & Studer, L. (2005)    PLoS Med 2, e161-   22. Amit, M., Carpenter, M. K., Inokuma, M. S., Chiu, C. P.,    Harris, C. P., Waknitz, M. A., Itskovitz-Eldor, J. &    Thomson, J. A. (2000) Dev Biol 227, 271-278.-   23. Montjovent, M. O., Burri, N., Mark, S., Federici, E., Scaletta,    C., Zambelli, P. Y., Hohlfeld, P., Leyvraz, P. F., Applegate, L. L.    & Pioletti, D. P. (2004) Bone 35, 1323-1333.-   24. Kim, M. S., Hwang, N. S., Lee, J., Kim, T. K., Leong, K.,    Shamblott, M. J., Gearhart, J. & Elisseeff, J. (2005) Stem Cells 23,    113-123.-   25. Hyllested, J. L., Veje, K. & Ostergaard, K. (2002)    Osteoarthritis Cartilage 10, 333-343.-   26. Ma, Z., Kotaki, M., Inai, R. & Ramakrishna S. (2005) Tissue Eng    11, 101-109.-   27. Maniatopoulos, C., Sodek, J. & Melcher, A. H. (1988) Cell Tissue    Res 254, 317-330.-   28. Coelho, M. J. & Fernandes, M. H. (2000) Biomaterials 21,    1095-1102.-   29. Smith, N., Dong, Y., Lian, J. B., Pratap, J., Kingsley, P. D.,    van Wijnen, A. J., Stein, J. L., Schwarz, E. M., O'Keefe, R. J.,    Stein, G. S. et al (2005) Cell Physiol 203, 133-143.-   30. Rossert, J., Terraz, C. & Dupont, S. (2000) Nephrol Dial    Transplant 15, Suppl. 66-88.-   31. Bellows, C. G., Aubin, J. E. & Heersche, J. N. (1991) Bone Miner    14, 27-40.-   32. Sodek, J., Ganss, B. & McKee, M. D. (2000) Crit Rev Oral Biol    Med 11,279-303-   33. Mannstadt, M., Juppner, H. & Gardella, T. J. (1999) Am J Physiol    277, F665-F675.-   34. Sperger, J. M., Chen, X., Draper, J. S., Antosiewicz, J. E.,    Chon, C. H., Jones, S. B., Brooks, J. D., Andrews, P. W.,    Brown, P. O. & Thomson, J. A. (2003) Proc Natl Acad Sci USA 100,    13350-13355.-   35. Golan-Mashiach, M., Dazard, J. E., Gerecht-Nir, S., Amariglio,    N., Fisher, T., Jacob-Hirsch, J., Bielorai, B., Osenberg, S., Barad,    O., Getz, G. et al (2005) FASEB J 19, 147-149.-   36. Bhattacharya, B., Miura, T., Brandenberger, R., Mejido, J., Luo,    Y., Yang, A. X., Joshi, B. H., Ginis, I., Thies, R. S., Amit, M. et    al (2004) Blood 103, 2956-2964.-   37. Zipori, d. (2004) Nat Rev Genet 5, 873-878.-   38. Karabela-Bouropoulou, V., Markaki, S. & Milas, C. (1988) Pathol    Res Pract 183, 761-766.-   39. Reichenberger, E., Aigner, T., von der Mark, K., Stoss, H. &    Bertling, W. (1991) Dev Biol 148, 562-572.-   40. Levine, J. M. & Nishiyama, A. (1996) Perspect Dev Neurobiol, 3,    245-259.-   41. Hedbom, E., Antonsson, P., Hjerpe, A., Aeschlimann, D.,    Paulsson, M., Rosa-Pimentel, E., Sommarin, Y., Wendel, M.,    Oldberg, A. & Heinegard, D. (1992) J Biol Chem 267, 6132-6136.-   42. Dvash, T. & Benvenisty, N. (2004) Best Pract Res Clin Obstet    Gynaecol 18, 929-940.-   43. Mistry, A. S. & Mikos, A. G. (2005) Adv Biochem Eng Biotechnol    94, 1-22.-   44. Noth, U., Osyczka, A. M., Tuli, R., Hickok, N. J.,    Danielson, K. G. & Tuan, R. S. (2002) J Orthop Res 20, 1060-1069.-   45. Aigner, T. & Stove, J. (2003) Adv Drug Deliv Rev 55, 1569-1593.-   46. Scott, J. E. (2003) J Physiol 553, 335-343.-   47. Cell Junctions, Cell Adhesion, and the Extracellular    Matrix (1994) in Molecular Biology of the Cell, eds. Alberts, B.,    Johnson, A., Lewis, J., Raff, M., Roberts, K. & Walter, P. (Garland    Publishing), pp. 971-995.-   48. Yoshimoto, H., Shin, Y. M., Terai, H. & Vacanti, J. P. (2003)    Biomaterials 24, 2077-2282.-   49. Canty, E. G. & Kadler, K. E. (2002) Comp Biochem Physiol A Mol    Integr Physiol 133, 979-985.-   50. Brent, A. E., Schweitzer, R. & Tabin, C. J. (2003) Cell 113,    235-248.-   51. Brent, A. E., Braun, T. & Tabin, C. J. (2005) Development 132,    515-528.-   52. Vunjak-Novakovic, G., Altman, G., Horan, R. &    Kaplan, D. L. (2004) Annu Rev Biomed Eng 6, 131-156.-   53. Van Eijk, F., Saris, D. B., Riesle, J., Willems, W. J., Van    Blitterswijk, C. A., Verbout, A. J. & Dhert, W. J. (2004) Tissue Eng    10, 893-903.-   54. Calve, S., Dennis, R. G., Kosnik, P. E. 2nd, Baar, K., Grosh, K.    & Arruda, E. M. (2004) Tissue Eng 10, 755-761.-   55. Grenier, G., Remy-Zolghadri, M., Larouche, D., Gauvin, R.,    Baker, K., Bergeron, F., Dupuis, D., Langelier, E., Rancourt, D.,    Auger, F. A. et al (2005) Tissue Eng 11, 90-100.-   56. Lee, C. H., Shin, H. J., Cho, I. H., Kang, Y. M., Kim, I. A.,    Park, K. D. & Shin, J. W. (2005) Biomaterials 26, 1261-1270.-   57. Cartmell, J. S. & Dunn, M. G. (2000) J Biomed Mater Res 49,    134-140.-   58. Wren, T. A., Lindsey, D. P., Beaupre, G. S. &    Carter, D. R. (2003) Ann Biomed Eng 31, 710-717.

What is claimed is:
 1. A method of generating a tendon in vitro,comprising: (a) culturing connective tissue progenitor cells in theabsence of a scaffold or a carrier to thereby obtain a sheet-liketissue, and (b) rolling and/or folding said sheet-like tissue to form arounded cylinder, thereby generating the tendon tissue in vitro.
 2. Themethod of claim 1, wherein said culturing is performed in a culturemedium which comprises ascorbic acid and/or dexamethasone.
 3. The methodof claim 1, wherein said culturing is effected without culturesplitting.
 4. The method of claim 3, wherein said culturing without saidculture splitting is effected for about 4-5 weeks.
 5. The method ofclaim 1, further comprising removing said sheet-like tissue from saidculturing vessel prior to said rolling and/or to said folding of saidsheet-like tissue.
 6. The method of claim 5, wherein said removing iseffected using a scraper.
 7. The method of claim 1, wherein saidconnective tissue progenitor cells are capable of differentiating intoat least two cell lineages of a connective tissue.
 8. The method ofclaim 7, wherein said at least two cell lineages of said connectivetissue are selected from the group consisting of an osteogenic lineage,a chondrogenic lineage, an adipocytic lineage and a tendon and ligamentlineage.
 9. The method of claim 1, wherein said culturing is effected ata cell density of about 5-10×10⁵ cells/cm².
 10. The method of claim 2,wherein said culture medium further comprises serum or serumreplacement.
 11. The method of claim 1, wherein said connective tissueprogenitor cells express CD105, CD166, CD44, CD29 and HLA-ABC.
 12. Themethod of claim 1, wherein said connective tissue progenitor cells notexpressing CD45 and HLA-DR.
 13. The method of claim 1, wherein saidconnective tissue progenitor cells express core binding factor alpha 1(CBFA1).
 14. The method of claim 1, wherein said connective tissueprogenitor cells are devoid of feeder cells.
 15. The method of claim 1,wherein said connective tissue progenitor cells are xeno-free.